Inhibition of cell proliferation

ABSTRACT

The disclosed modulators of Rb:Raf-1 interactions are potent, selective disruptors of Rb:Raf-1 binding, with IC 50  values ranging from 80 nM to 500 nM. Further, these compounds are surprisingly effective in inhibiting a wide variety of cancer cells, including osteosarcoma, epithelial lung carcinoma, non-small cell lung carcinoma, three different pancreatic cancer cell lines, two different glioblastoma cell lines, metastatic breast cancer, melanoma, and prostate cancer. Moreover, the disclosed compounds effectively disrupt angiogenesis and significantly inhibited tumors in nude mice derived from human epithelial lung carcinoma tumors. Accordingly, the disclosed compounds, pharmaceutical compositions comprising the compounds, methods of inhibiting cell proliferation, methods of treating subjects with cancer, and methods of preparing the disclosed compounds are provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication Ser. No. 60/738,776, filed Nov. 22, 2005, the entireteachings of which are incorporated herein by reference.

GOVERNMENT SUPPORT

This invention was made with government support under 5R01CA063136-14awarded by the National Cancer Institute. The government has certainrights in the invention.

TECHNICAL FIELD

This application relates to compounds, pharmaceutical compositions, andmethods for modulating the Rb:Raf-1 interaction in vitro or in vivo, andmore particularly to treatment of disorders modulated by the Rb:Raf-1interaction, for example, proliferation disorders such as cancer.

BACKGROUND

The inactivation of the retinoblastoma tumor suppressor protein Rb bycell cycle regulatory kinases is disrupted in almost all cancers. Innormal cells, inactivation of Rb is necessary for the G1 to S phaseprogression of the cell cycle. Rb controls entry into the S phase byrepressing the transcriptional activity of the E2F family oftranscription factors, especially E2Fs 1, 2, and 3. Rb is inactivatedthrough multiple phosphorylation events mediated by kinases associatedwith D and E type cyclins in the G1 phase of the cell cycle. It wasfound that the signaling kinase Raf-1 initiates the phosphorylationevents; Raf-1 signaling kinase is known to play a role in promotingcancer, and studies have shown that Rb:Raf-1 binding facilitates cellproliferation. It has also been found that the Rb:Raf-1 interaction iselevated in human tumors compared to adjacent normal tissue in 80% ofsamples examined. Because Raf-1 is persistently activated in manytumors, a few attempts have been made to selectively inhibit tumors bymodulating Rb and/or Raf-1 activity with Raf-1 antisenseoligonucleotides, the multikinase inhibitor Sorafenib, and a peptidefragment of Raf-1 coupled to a carrier peptide. However, there is stilla need for effective modulators of the Rb:Raf-1 interaction.

SUMMARY

Applicants have discovered modulators of Rb:Raf-1 interactions that aresurprisingly effective in inhibiting the tumor growth and survival of awide variety of cancer cells. For example, certain disclosed compoundsare potent, selective disruptors of Rb:Raf-1 binding, with IC₅₀ valuesranging from 80 nM to 500 nM (Examples 5-8). Also, the disclosedcompounds are surprisingly effective in inhibiting the tumor growth andsurvival of a wide variety of cancer cells, including osteosarcoma(Example 9), epithelial lung carcinoma (Example 10), non-small cell lungcarcinoma (Example 11), three different pancreatic cancer cell lines(Example 12), two different glioblastoma cell lines (Example 13),metastatic breast cancer (Example 14), melanoma (Example 15), andprostate cancer (Example 16). Moreover, the disclosed compoundseffectively disrupt angiogenesis (Example 18), significantly inhibitedanchorage independent tumor growth (Example 19) and significantlyinhibited the growth of human epithelial lung carcinoma in nude mice(Example 20). Accordingly, compounds, pharmaceutical compositionscomprising the compounds, methods of inhibiting cell proliferation,methods of treating subjects with cancer, and methods of preparing thedisclosed compounds are provided herein.

The compound is represented by structural formula Ia:

and pharmaceutically acceptable salts and solvates thereof.

In the compound represented by structural formula Ia, each dashed line(- - -) represents a single bond, or one dashed line (- - -) is a doublebond. Typically, one dashed line is a double bond and the other dashedlines are single bonds.

In the compound represented by structural formula Ia, Ring A isoptionally substituted. Zero, one, or two of the ring atoms in Ring Aare N. Further, Ring A is fused with zero, one, or two optionallysubstituted 3-15 membered monocyclic or polycyclic rings selected fromthe group consisting of aryl, heteroaryl, heterocyclyl, andcycloaliphatic. For example, in some embodiments, the ring representedby Ring A, together with any rings to which it can be fused, is anoptionally substituted phenyl, biphenyl, naphthyl, pyrenyl, anthracyl,9,10-dihydroanthracyl, fluorenyl, pyridyl, pyrimidyl, pyrazinyl,triazinyl, quinolinyl, isoquinolinyl, quinazolinyl, napthyridyl,pyridopyrimidyl, benzothienyl, benzothiazolyl, benzoisothiazolyl,thienopyridyl, thiazolopyridyl, isothiazolopyridyl, benzofuranyl,benzooxazolyl, benzoisooxazolyl, furanopyridyl, oxazolopyridyl,isooxazolopyridyl, indolyl, isoindolyl, benzimidazolyl, benzopyrazolyl,pyrrolopyridyl, isopyrrolopyridyl, imidazopyridyl, or pyrazolopyridylgroup. In certain embodiments, the ring represented by Ring A, togetherwith any rings to which it can be fused, is an optionally substitutedphenyl, naphthyl, anthracyl, fluorenyl, 9,10-dihydroanthracyl, pyridyl,pyrimidyl, pyrazinyl, triazinyl, quinolinyl, isoquinolinyl,quinazolinyl, or napthyridyl group. In particular embodiments, the ringrepresented by Ring A, together with any rings to which it can be fused,is an optionally substituted naphthyl, pyridyl, quinolinyl, orisoquinolinyl group, or a substituted phenyl group.

In some embodiments, one, two or three substitutable carbons in the ringrepresented by Ring A can be substituted with —F, —Cl, —Br, —I, —CN,—NO₂, C₁₋₆ alkyl, C₁₋₆ alkoxy, —CF₃, or C₁₋₆ haloalkoxy, or twosubstitutable carbons in the ring represented by Ring A are linked withC₁₋₂ alkylenedioxy. In some embodiments, at least one ring atom of RingA adjacent to the point of attachment of Ring A to the rest of thecompound is unsubstituted, for example, when Ring A is phenyl and Y isat the 1-position, either the 2 or the 6 position is unsubstituted.

In some embodiments, Ring A can be a six-membered ring that ismonsubstituted at its 2, 3, or 4 positions or disubstituted at its 2,3,2,4, 2,5 or 3,4 positions with substituents independently selected fromthe group consisting of —F, —Cl, —Br, —NO₂, C₁₋₆ alkyl, —CF₃, andmethylenedioxy, where the 1 position is the point of attachment of RingA to the rest of the compound, e.g., the point of attachment of Ring Ato Y. In some embodiments, Ring A is a phenyl independentlydisubstituted with —Br, —Cl, —F, or —CF₃ at the 2,3, 2,4, or 2,5positions of Ring A, where the 1 position is the point of attachment ofRing A to the rest of the compound.

In the compound represented by structural formula Ia, Y is an optionallysubstituted C₁₋₃ alkylene or C₁₋₃ alkenylene linking group. In someembodiments, Y can be optionally substituted with —OH, C₁₋₆ alkyl, C₁₋₆alkoxy, C₁₋₆ haloalkyl, C₁₋₆ haloalkoxy, or C₁₋₆ aralkyl, ═O, or ═S.Generally, Y is optionally substituted with —OH, C₁₋₆ alkyl, C₁₋₆alkoxy, C₁₋₆ aralkyl, ═O, or ═S. Preferably, Y is unsubstituted orsubstituted with C₁₋₃ alkyl. Typically, Y can be ethylene or methylene.In certain embodiments, Y is unsubstituted or substituted with C₁₋₃alkyl. When a substituent, is bonded to Y, such a substitutent may bebound to any carbon in Y. Typically, a substituent can be bonded to thecarbon in Y adjacent to Ring A.

In the compound represented by structural formula Ia, X¹ isindependently —O—, —S—, or optionally substituted —CH₂—, —CH═, —NH—, or—N═, and X² and X³ are independently ═S, or optionally substituted —NH₂,═NH, or —SH, or an optionally substituted 3-7 membered aryl, heteroaryl,heterocyclyl, or cycloaliphatic ring. Or, X² and X³ are independently—S—, or optionally substituted —NH—, —N═, or ═N—, and X² and X³ arelinked α, β, or γ through an optionally substituted alkyl, alkenyl,heteroalkyl, heteroalkenyl, heteroatom, aryl, heteroaryl, heterocyclyl,or cycloaliphatic linking group, thereby forming an optionallysubstituted heteroaryl or heterocyclyl ring. Or, X² is independently —S—or optionally substituted —NH—, —N═, or ═N—, and X² is linked α, β, or γto a carbon of Y through an optionally substituted alkyl, alkenyl,heteroalkyl, heteroalkenyl, or heteroatom linking group, thereby formingan optionally substituted heteroaryl or heterocyclyl ring, and whereinX³ is optionally —H, and wherein X³ is optionally —H. In someembodiments, X³ is not —H.

Each substitutable carbon in structural formula Ia and the variablesrepresented in structural formula Ia can be optionally substituted witha carbon substituent independently selected from the group consisting of—F, —Cl, —Br, —I, —CN, —NO₂, —R^(a), —OR^(a), —C(O)R^(a), —OC(O)R^(a),—C(O)OR^(a), —SR^(a), —C(S)R^(a), —OC(S)R^(a), —C(S)OR^(a), —C(O)SR^(a),—C(S)SR^(a), —S(O)R^(a), —SO₂R^(a), —SO₃R^(a), —OSO₂R^(a), —OSO₃R^(a),—PO₂R^(a)R^(b), —OPO₂R^(a)R^(b), —PO₃R^(a)R^(b), —OPO₃R^(a)R^(b),—N(R^(a)R^(b)), —C(O)N(R^(a)R^(b)), —C(O)NR^(a)NR^(b)SO₂R^(c),—C(O)NR^(a)SO₂R^(c), —C(O)NR^(a)CN, —SO₂N(R^(a)R^(b)), —NR^(a)SO₂R^(b),—NR^(c)C(O)R^(a), —NR^(c)C(O)OR^(a), —NR^(c)C(O)N(R^(a)R^(b)),—C(NR^(c))—N(R^(a)R^(b)), —NR^(d)—C(NR^(c))—N(R^(a)R^(b)),NR^(a)N(R^(a)R^(b)), —CR^(c)═CR^(a)R^(b), —C≡CR^(a), ═O, ═S,═CR^(a)R^(b), ═NR^(a), ═NOR^(a), and ═NNR^(a), or two substitutablecarbons can be linked with C₁₋₃ alkylenedioxy. In some embodiments, eachsubstitutable carbon can be optionally substituted with —F, —Cl, —Br,—I, —CN, —NO₂, —R^(a), —OR^(a), —C(O)R^(a), —OC(O)R^(a), —C(O)OR^(a),—SR^(a), —SO₂R^(a), —SO₃R^(a), —OSO₂R^(a), —OSO₃R^(a), —N(R^(a)R^(b)),—C(O)N(R^(a)R^(b)), —C(O)NR^(a)NR^(b)SO₂R^(c), —C(O)NR^(a)SO₂R^(c),—SO₂N(R^(a)R^(b)), —NR^(a)SO₂R^(b), —NR^(c)C(O)R^(a), —NR^(c)C(O)OR^(a),═S, or ═O, or two substitutable carbons can be linked with C₁₋₃alkylenedioxy.

Each substitutable nitrogen in structural formula Ia and the variablesrepresented in structural formula Ia can optionally substituted with anitrogen substituent independently selected from the group consisting of—CN, —NO₂, —R^(a), —OR^(a), —C(O)R^(a), —C(O)R^(a)-aryl, —OC(O)R^(a),—C(O)OR^(a), —SR^(a), —S(O)R^(a), —SO₂R^(a), —SO₃R^(a), —N(R^(a)R^(b)),—C(O)N(R^(a)R^(b)), —C(O)NR^(a)NR^(b)SO₂R^(c), —C(O)NR^(a)SO₂R^(c),—C(O)NR^(a)CN, —SO₂N(R^(a)R^(b)), —NR^(a)SO₂R^(b), —NR^(c)C(O)R^(a),—NR^(c)C(O)OR^(a), —NR^(c)C(O)N(R^(a)R^(b)), and oxygen to form anN-oxide. Further, each substitutable nitrogen in structural formula Iaand the variables represented in structural formula Ia can be optionallyprotonated or quaternary substituted to carry a positive charge, whichcan be balanced by a pharmaceutically acceptable counteriion.

Substitutable carbons or nitrogens in structural formula Ia include thecarbons in Ring A which are not bound to Y, or the nitrogens whichreplace one or two carbons in Ring A. Further, examples of substitutablecarbons or nitrogens in the variables represented in structural formulaIa include, e.g., the carbons in linker Y, the carbons in X¹ when X¹ is—CH₂— or —CH═, the nitrogens in X¹ when X¹ is —NH— or —N═, the nitrogenswhen X² and X³ are —NH₂, ═NH, substitutable carbons in the 3-7 memberedaryl, heteroaryl, heterocyclyl, or cycloaliphatic rings which can beselected for X² and X³, and the like.

In the compound represented by structural formula Ia, each R^(a)-R^(d)is independently —H, C₁₋₆ alkyl, C₁₋₆ alkoxy, C₁₋₆ haloalkyl, C₁₋₆haloalkoxy, C₁₋₆ aralkyl, aryl, heteroaryl, heterocyclyl, orcycloaliphatic, or, —N(R^(a)R^(b)), taken together, is an optionallysubstituted heterocyclic group. In some embodiments, each R^(a)-R^(d) isindependently —H, C₁₋₆ alkyl, C₁₋₆ alkoxy, C₁₋₆ haloalkyl, C₁₋₆haloalkoxy, C₁₋₆ aralkyl, aryl, heteroaryl, heterocyclyl, orcycloaliphatic. In some embodiments, each R^(a)-R^(d) is independently—H or C₁₋₆ alkyl.

In the compound represented by structural formula Ia, one or moreprotecting groups may optionally protect one or more of X², X³, eachnitrogen substitutent, and each carbon substituent.

In some embodiments, the compound represented by structural formula Iadoes not include benzylN,N′-bis(tert-butoxycarbonyloxy)carbamimidothioate. In some embodiments,the compound represented by structural formula Ia is not benzylcarbamimidothioate.

In some embodiments, the compound represented by structural formula Iadoes not include the 3,4,6-trichloro-2-nitrophenolate salt of2,4-dichlorobenzyl carbamimidothioate or the 2,4-dinitrophenolate saltof (4-chloronaphthalen-1-yl)methyl carbamimidothioate:

In certain embodiments, the compound is not 2,4-dichlorobenzylcarbamimidothioate or (4-chloronaphthalen-1-yl)methylcarbamimidothioate. In particular embodiments, the compounds representedby structural formula Ia do not include 3,4,6-trichloro-2-nitrophenolateor 2,4-dinitrophenolate salts.

In some embodiments, Ring A and Y, taken together, are not anunsubstituted benzyl group. In certain embodiments, Ring A is not anunsubstituted phenyl group. In some embodiments, X¹, X², and X³, takentogether with the carbon atom between them are not an unsubstitutedguanidinyl group. Likewise, in some embodiments, the second reagent isnot unsubstituted guanidine. In some embodiments, X¹, X², and X³, takentogether with the carbon atom between them are not an unsubstitutedthioureayl group where X¹ is S. In certain embodiments, X¹, X², and X³,taken together with the carbon atom between them are not anunsubstituted thioureayl group. Likewise, in some embodiments, thesecond reagent is not unsubstituted thiourea. In some embodiments, X¹,X², and X³, taken together with the carbon atom between them are not anunsubstituted ureayl group where X¹ is O. In certain embodiments, X¹,X², and X³, taken together with the carbon atom between them are not anunsubstituted ureayl group. Likewise, in some embodiments, the secondreagent is not unsubstituted urea.

In some embodiments, the compound is represented by structural formulaIb:

The variables in structural formula Ib can take any values as describedfor those variables herein. In some embodiments, in the compoundrepresented by structural formula Ib, R^(y) is —H, —OH, C₁₋₆ alkyl, C₁₋₆alkoxy, C₁₋₆ haloalkyl, C₁₋₆ haloalkoxy, or C₁₋₆ aralkyl, or —R^(y) is═O or ═S. X¹ and X³ are independently N or S. Z¹ is a heteroatom or a β-or γ-bonded alkyl, alkenyl, heteroalkyl, heteroalkenyl, aryl,heteroaryl, heterocyclyl, or cycloaliphatic linking group, whereby RingB is a heterocyclyl or heteroaryl ring. Ring B is optionally substitutedat any substitutable ring atom with halogen, —CN, —NO₂, —R^(a), —OR^(a),—C(O)R^(a), —OC(O)R^(a), —C(O)OR^(a), —SR^(a), —SO₂R^(a), —SO₃R^(a),—OSO₂R^(a), —OSO₃R^(a), —N(R^(a)R^(b)), —C(O)N(R^(a)R^(b)),—SO₂N(R^(a)R^(b)), —NR^(a)SO₂R^(b), —NR^(c)C(O)R^(a), —NR^(c)C(O)OR^(a),or ═O.

In some embodiments, the compound is represented by structural formulaIc:

The variables in structural formula Ic can take any values as describedfor those variables herein. In some embodiments, in the compoundrepresented by structural formula Ic, Z¹ is 1,2-phenylene, —CH₂CH₂—,—CH═CH—, or —N═CH₂—; and Ring B is optionally substituted at anysubstitutable ring atom with halogen, —CN, —NO₂, —R^(a), —OR^(a),—N(R^(a)R^(b)), —C(O)N(R^(a)R^(b)), —NR^(c)C(O)R^(a), or ═O. In someembodiments, Z¹ is —N═CH₂— optionally substituted with —N(R^(a)R^(b)).In certain embodiments, the compound, e.g., represented by structuralformula Ic, is selected from the group consisting of:

In some embodiments, the compound is represented by structural formulaId:

The variables in structural formula Id can take any values as describedfor those variables herein. In some embodiments, in the compoundrepresented by structural formula Id, R³, R³′, R^(y) and R² areindependently —H, —OH, C₁₋₆ alkyl, C₁₋₆ alkoxy, C₁₋₆ haloalkyl, C₁₋₆haloalkoxy, C₁₋₆ aralkyl, aryl, heteroaryl, heterocyclyl, orcycloaliphatic, or —R^(y) is ═O or ═S. In some embodiments, R^(y) is —H,C₁₋₆ alkyl, C₁₋₆ aralkyl, or ═O. In certain embodiments, the compound,e.g., represented by structural formula Id, is selected from the groupconsisting of:

In certain embodiments, the compound, e.g., represented by structuralformula Id, is selected from the group consisting of:

In certain embodiments, the compound, e.g., represented by structuralformula Id, is selected from the group consisting of:

In some embodiments, the compound is represented by structural formulaIe:

The variables in structural formula Ie can take any values as describedfor those variables herein. In some embodiments, in the compoundrepresented by structural formula Ie, R¹ is —H, —OH, C₁₋₆ alkyl, C₁₋₆alkoxy, C₁₋₆ haloalkyl, C₁₋₆ haloalkoxy, C₁₋₆ aralkyl, aryl, heteroaryl,heterocyclyl, or cycloaliphatic, or —R¹ represents a lone pair of thenitrogen to which it is attached.

In some embodiments, the compound is represented by structural formulaIf:

The variables in structural formula If can take any values as describedfor those variables herein. In some embodiments, in the compoundrepresented by structural formula If, R³, R³′, R¹ and R² areindependently —H, —OH, C₁₋₆ alkyl, C₁₋₆ alkoxy, C₁₋₆ haloalkyl, C₁₋₆haloalkoxy, C₁₋₆ aralkyl, aryl, heteroaryl, heterocyclyl, orcycloaliphatic, or —R¹ is ═O or ═S. In some embodiments, R^(y) is —H,C₁₋₆ alkyl, or C₁₋₆ aralkyl, or —R^(y) is ═O. In certain embodiments,the compound, e.g., represented by structural formula If, is selectedfrom the group consisting of:

In some embodiments, the compound is represented by structural formulaIg:

The variables in structural formula Ie can take any values as describedfor those variables herein. In some embodiments, in the compoundrepresented by structural formula Ie, X³ is —NR³R³′ or optionallyhalogenated phenyl; R³, R³′, and R¹ are independently —H, C₁₋₆ alkyl,C₁₋₆ alkanoyl or C₁₋₆ aralkanoyl, or —R¹ represents a lone pair of thenitrogen to which it is attached; Z² is a β-bonded alkyl or alkenyllinking group, whereby Ring C is a heterocyclyl or heteroaryl ring; andRing C is optionally substituted at any substitutable ring atom withhalogen, —CN, —NO₂, —R^(a), —OR^(a), —N(R^(a)R^(b)), ═O, or phenyl. Incertain embodiments, the compound, e.g., represented by structuralformula Ig, is selected from the group consisting of:

In certain embodiments, the compound, e.g., represented by structuralformula Ig, is selected from the group consisting of:

In some embodiments, the compound is represented by structural formulaIh:

The variables in structural formula Ih can take any values as describedfor those variables herein. In some embodiments, in the compoundrepresented by structural formula Ih, R³, R³′, R¹ and R² areindependently —H, —OH, C₁₋₆ alkyl, C₁₋₆ alkoxy, or C₁₋₆ aralkyl,provided that at least two of R³, R³′, R^(y) and R² are —H; or R³ and R²together are —N═CH— or —CH═N—, optionally substituted with—N(R^(a)R^(b)) or —C(O)N(R^(a)R^(b)). The ring represented by Ring A isan optionally substituted naphthyl, pyridyl, quinolinyl, orisoquinolinyl; or a phenyl that is monsubstituted at its 2, 3, or 4positions or disubstituted at its 2,3, 2,4, or 2,5 or 3,4 positions,wherein the 1 position is the point of attachment of Ring A to the restof the compound. Typically, substituents for Ring A are independently—Br, —Cl, —F, —CN, —NO₂, C₁₋₆ alkoxy, methylenedioxy or —CF₃, providedthat when Ring A is phenyl at least one substituent of Ring A is —F or—Cl.

A method of synthesizing a compound represented by structural formula Iaincludes reacting a first reagent represented by structural formula IIawith a second reagent represented by structural formula IIIa, therebyforming the a compound represented by structural formula Ia:

In the above structural formulas, each dashed line (- - -) represents asingle bond, or one (- - -) is a double bond.

Y and Y′ are an optionally substituted C₁₋₃ alkylene or C₁₋₃ alkenylene.

X¹′ is independently ═CH₂, —NH₂, ═NH, —OH, —SH, ═O, or ═S, and X¹ iscorrespondingly —O—, —S—, or optionally substituted —CH₂—, —CH═, —NH—,or —N═, wherein when W is unsubstituted —NH₂, X¹′ is and X¹ isoptionally substituted —NH— or —N═. X²′ and X³′ are independently ═S oroptionally substituted —NH₂, ═NH, or —SR^(a), or an optionallysubstituted 3-7 membered aryl, heteroaryl, heterocyclyl, orcycloaliphatic ring, and X² and X³ correspond to X²′ and X³′,respectively, or X²′ and X³′ are independently —S— or optionallysubstituted —NH—, —N═, or ═N—, and are optionally linked α, β, or γthrough an optionally substituted alkyl, alkenyl, heteroalkyl,heteroalkenyl, heteroatom, aryl, heteroaryl, heterocyclyl, orcycloaliphatic linking group, thereby forming an optionally substitutedheteroaryl or heterocyclyl ring, wherein X² and X³ are likewised linkedto correspond to X²′ and X³′, respectively; or X²′ is independently ═Sor optionally substituted —NH₂, ═NH, or —SH, X² is correspondingly —S—or optionally substituted —NH—, —N═, or ═N—, and X² is linked α, β, or γto a carbon of Y through an optionally substituted alkyl, alkenyl,heteroalkyl, heteroalkenyl, or heteroatom linking group, thereby formingan optionally substituted heteroaryl or heterocyclyl ring, and whereinX³ is optionally —H. In some embodiments, X³ is not —H. In someembodiments, X¹′ is —OH, ═O, —SH, or ═S, and X¹ is correspondingly —O—or —S—. In some embodiments, X¹′ is —SH or ═S and X¹ is correspondingly—S—.

W is a leaving group or unsubstituted —NH₂. The variable W can representany leaving group known to the art. For example, in some embodiments, Wcan be halogen, perchlorate, amonioalkanesulfonate, halosulfonate,haloalkyl sulfonate, alkyl sulfonate, optionally substituted arylsulfonate, or optionally substituted —N(arylsulfonate)₂. W can also beoptionally alkylated halonium ion or optionally alkylated oxonium ion,prepared in situ. In some embodiments, W is —Cl, —Br, —I, —OSO₂F,—OSO₂CF₃, —OSO₂C₄F₉, —OSO₂CH₂CF₃, —OSO₂CH₃, tosylate, brosylate,nosylate, or —N(tosylate)₂. In certain embodiments, W is —Cl or —Br.

Each substitutable carbon and nitrogen is optionally substituted asdescribed herein above for any of structural formulas Ia-Ih.

X²′, X³′, X², X³, each nitrogen substituent, and each carbon substituentare each optionally protected with a protecting group as describedherein above for structural formula Ia.

The method is provided that the compound is not benzylN,N′-bis(tert-butoxy carbonyloxy) carbamimidothioate or a saltrepresented by

In various embodiments of the method, the first and second reagents arereacted together under microwave irradiation.

In some embodiments, the first reagent is represented by structuralformula IIb and the second reagent is represented by structural formulaIIIb, whereby the compound produced is represented by structural formulaIb:

In the above structural formulas Ib, IIb and IIIb, R^(y) and R^(y)′ are—H, —OH, C₁₋₆ alkyl, C₁₋₆ alkoxy, C₁₋₆ haloalkyl, C₁₋₆ haloalkoxy, orC₁₋₆ aralkyl, or —R^(y) and —R^(y′) are ═O or ═S; X³′ and X³ are N or S;X¹′ is —OH or —SH and X¹ is correspondingly —O— or —S—; Z¹ and Z¹′ areeach a heteroatom or a β- or γ-bonded alkyl, alkenyl, heteroalkyl,heteroalkenyl, aryl, heteroaryl, heterocyclyl, or cycloaliphatic linkinggroup, whereby Ring B and Ring B′ are corresponding heterocyclyl orheteroaryl rings; and Ring B and Ring B′ are optionally substituted atany substitutable ring atom with halogen, —CN, —NO₂, —R^(a), —OR^(a),—C(O)R^(a), —OC(O)R^(a), —C(O)OR^(a), —SR^(a), —SO₂R^(a), —SO₃R^(a),—OSO₂R^(a), —OSO₃R^(a), —N(R^(a)R^(b)), —C(O)N(R^(a)R^(b)),—SO₂N(R^(a)R^(b)), —NR^(a)SO₂R^(b), —NR^(c)C(O)R^(a), —NR^(c)C(O)OR^(a),or ═O. In some embodiments, X¹′ is —SH and X¹ is correspondingly —S—. Insome embodiments, Z¹ and Z¹′ are each 1,2-phenylene, —CH₂CH₂—, —CH═CH—,or —N═CH₂—; and Ring B and Ring B′ are optionally substituted at anysubstitutable ring atom with halogen, —CN, —NO₂, —R^(a), —OR^(a),—N(R^(a)R^(b)), —C(O)N(R^(a)R^(b)), —NR^(c)C(O)R^(a) or ═O. In someembodiments, Z¹ and Z¹′ are —N═CH₂— optionally substituted with—N(R^(a)R^(b)). In certain embodiments, the compound produced using thefirst reagent represented by IIb and the second reagent represented bystructural formula IIIb is selected from the group of specific compoundsshown above under structural formula Ib.

In some embodiments, the second reagent is represented by structuralformula IIIc, whereby the compound produced is represented by structuralformula Id:

In the above structural formulas IIIc and Id, R³, R³′, R^(y) and R² areindependently —H, —OH, C₁₋₆ alkyl, C₁₋₆ alkoxy, C₁₋₆ haloalkyl, C₁₋₆haloalkoxy, C₁₋₆ aralkyl, aryl, heteroaryl, heterocyclyl, orcycloaliphatic, or —R^(y) is ═O or ═S. In some embodiments, R^(y) is —H,C₁₋₆ alkyl, C₁₋₆ aralkyl, or ═O. In certain embodiments, the compoundproduced is selected from the group of specific compounds shown aboveunder by structural formula Id.

In some embodiments of the method, the first reagent is represented bystructural formula IIc, whereby the compound is represented bystructural formula Ie:

In the above structural formulas IIc and Ie, X¹′ is; none of X²′, X³′X², or X³ is unsubstituted —NH₂ or ═NH; R¹ is —H, —OH, C₁₋₆ alkyl, C₁₋₆alkoxy, C₁₋₆ haloalkyl, C₁₋₆ haloalkoxy, C₁₋₆ aralkyl, aryl, heteroaryl,heterocyclyl, or cycloaliphatic, or —R¹ represents a lone pair of thenitrogen to which it is attached; and one dashed line (- - -) representsa double bond and the other dashed lines represent single bonds.

In some embodiments, the second reagent is represented by structuralformula IIId, thereby producing a protected intermediate represented bystructural formula II:

In the above structural formulas IIId and Ii, R¹ is —H, —OH, C₁₋₆ alkyl,C₁₋₆ alkoxy, C₁₋₆ haloalkyl, C₁₋₆ haloalkoxy, C₁₋₆ aralkyl, aryl,heteroaryl, heterocyclyl, or cycloaliphatic, or —R¹ represents a lonepair of the nitrogen to which it is attached. PG and PG′ are amineprotecting groups. The protecting groups PG and PG′ can independently beany suitable amine protecting group, for example t-butyloxycarbonyl orbenzyloxycarbonyl.

In some embodiments, the first reagent and the second reagent arereacted together in the presence of an optionally substituted1-alkyl-2-halopyridinium salt or an optionally substituted1-aryl-2-halopyridinium salt. In certain embodiments, the first reagentand the second reagent are reacted together in the presence of1-methyl-2-chloropyridinium halide or 1-phenyl-2-chloropyridiniumhalide, typically 1-methyl-2-chloropyridinium iodide.

The method can further include removing protecting groups PG and PG′from the protected intermediate, thereby producing the compound,represented by structural formula Ij:

In some embodiments, when PG and PG′ are t-butyloxycarbonyl orbenzyloxycarbonyl, the step of removing PG and PG′ from the protectedintermediate can include contacting the protected intermediate withtrifluoracetic acid or SnCl₄. In certain embodiments, the compoundproduced is selected from the group of specific compounds shown aboveunder structural formula Ij.

In some embodiments, the first reagent is represented by structuralformula IId, and the second reagent is represented by structural formulaIIIe, whereby the compound is represented by structural formula Ig:

In the above structural formulas IId, IIIe, and Ig, Y′ is methylene or abond; X³ is —NR³R³′ or optionally halogenated phenyl; R³, R³′, and R¹are independently —H, C₁₋₆ alkyl, C₁₋₆ alkanoyl or C₁₋₆ aralkanoyl, or—R¹ represents a lone pair of the nitrogen to which it is attached; Z²is a β-bonded alkyl or alkenyl linking group, whereby Ring C is aheterocyclyl or heteroaryl ring; and Ring C is optionally substituted atany substitutable ring atom with halogen, —CN, —NO₂, —R^(a), —OR^(a),—N(R^(a)R^(b)), ═O, or phenyl. In certain embodiments, the compoundproduced is selected from the group of specific compounds shown aboveunder structural formula Ig.

In some embodiments, the first reagent is represented by structuralformula IIe, and the second reagent is represented by structural formulaIIIf, whereby the compound is represented by structural formula Ih:

In the above structural formulas IIe, IIIf, and Ih, X¹′ is —SH and X²′is ═NR², or X¹′ is ═S and X²′ is —NHR²; W′ is —Cl, —Br, or —I; and R³,R³′, R^(y) and R² are independently —H, —OH, C₁₋₆ alkyl, C₁₋₆ alkoxy, orC₁₋₆ aralkyl, provided that at least two of R³, R³′, R^(y) and R² are—H; or R³ and R² together are —N═CH— or —CH═N—, optionally substitutedwith —N(R^(a)R^(b)) or —C(O)N(R^(a)R^(b)).

The ring represented by Ring A is an optionally substituted naphthyl,pyridyl, quinolinyl, or isoquinolinyl; or a phenyl that ismonsubstituted at its 2, 3, or 4 positions or disubstituted at its 2,3,2,4, or 2,5 or 3,4 positions, wherein the 1 position is the point ofattachment of Ring A to the rest of the compound. Substituents for RingA are independently —Br, —Cl, —F, —CN, —NO₂, C₁₋₆ alkoxy, methylenedioxyor —CF₃, provided that when Ring A is phenyl at least one substituent ofRing A is —F or —Cl.

In some embodiments, the first reagent is prepared from a third reagentrepresented by structural formula IV:

by converting the hydroxyl group bound to Y in the third reagent to thegroups represented by W such as —NH₂ or a leaving group selected fromhalogen, optionally alkylated halonium ion, optionally alkylated oxoniumion, perchlorate, amonioalkanesulfonate, halosulfonate, haloalkylsulfonate, alkyl sulfonate, optionally substituted aryl sulfonate, oroptionally substituted —N(arylsulfonate)₂.

In some embodiments, the third reagent is prepared by reacting a fourthreagent represented by structural formula Va:

with R^(y)′MgCl, R^(y)′MgBr, R^(y)′MgI, R^(y)′Li, R^(y)′Na, or R^(y)′K.Such derivatives can be readily prepared from the correspondingR^(y)′-halide.

In some embodiments, the third reagent can be prepared by reducing afifth reagent represented by structural formula Vb:

Typical reagents and conditions for reducing carbonyls to alcohols arewell-known, for example, lithium aluminum hydride, sodium borohydride,lithium hydride, sodium hydride, potassium hydride, hydrogen in thepresence of a catalyst, e.g., Pd or Pt on carbon, electrochemicalmethods, and the like.

A pharmaceutical composition includes a compound represented bystructural formulas Ia-Ih and optionally a pharmaceutically acceptablecarrier or excipient.

A protein:ligand complex includes a compound represented by structuralformula Ia and at least one protein selected from the group consistingof retinoblastoma tumor suppressor protein and serine-threonine kinaseRaf-1. The complex can include a disclosed compound, retinoblastomatumor suppressor protein, and serine-threonine kinase Raf-1.

Various methods of treatment of cells and subjects are included. Forexample, a method of inhibiting proliferation of a cell includescontacting the cell with an effective amount of the disclosed compoundsor compositions. Typically, regulation of proliferation in the cell ismediated by at least one protein selected from the group consisting ofretinoblastoma tumor suppressor protein and serine-threonine kinaseRaf-1. For example, in various embodiments, the cells have an elevatedlevel of Rb, Raf-1, or Rb bound to Raf-1. In some embodiment, the methodincludes assaying the level of Rb, Raf-1, or Rb bound to Raf-1 in thecell.

A method of modulating the Rb:Raf-1 interaction in a proliferating cellincludes contacting the cell with an effective amount of the disclosedcompounds or compositions.

A method of modulating the Rb:Raf-1 interaction in a proliferating cellincludes contacting the cell with a modulator of the Rb:Raf-1interaction that is suitable for oral administration. In someembodiments, the modulator of the Rb:Raf-1 interaction is orallyadministered.

A method of treating or ameliorating a cell proliferation disorderincludes contacting the proliferating cells with an effective amount ofthe disclosed compounds or compositions Typically, regulation of cellproliferation in the disorder can be mediated by at least one proteinselected from the group consisting of retinoblastoma tumor suppressorprotein and serine-threonine kinase Raf-1. In some embodiments, theregulation of proliferation in the cells is mediated by the interactionbetween retinoblastoma tumor suppressor protein and serine-threoninekinase Raf-1. In various embodiments, the cell proliferation disorder iscancer or a non-cancerous cell proliferation disorder. In someembodiments, the cell proliferation disorder includes angiogenesis orthe cell proliferation disorder is mediated by angiogenesis.

In various embodiments, the cell proliferation disorder is or theproliferating cells are derived from a cancererous or a non-cancerouscell proliferation disorder. Exemplary cancererous and non-cancerouscell proliferation disorders include fibrosarcoma, myxosarcoma,liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer,ovarian cancer, prostate cancer, squamous cell carcinoma, basal cellcarcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous glandcarcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testiculartumor, lung carcinoma, small cell lung carcinoma, non-small cell lungcarcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma,melanoma, neuroblastoma, retinoblastoma, acute lymphocytic leukemia,lymphocytic leukemia, large granular lymphocytic leukemia, acutemyelocytic leukemia, chronic leukemia, polycythemia vera, Hodgkin'slymphoma, non-Hodgkin's lymphoma, multiple myeloma, Waldenstrobm'smacroglobulinemia, heavy chain disease, lymphoblastic leukemia, T-cellleukemia, T-lymphocytic leukemia, T-lymphoblastic leukemia, B cellleukemia, B-lymphocytic leukemia, mixed cell leukemias, myeloidleukemias, myelocytic leukemia, myelogenous leukemia, neutrophilicleukemia, eosinophilic leukemia, monocytic leukemia, myelomonocyticleukemia, Naegeli-type myeloid leukemia, nonlymphocytic leukemia,osteosarcoma, promyelocytic leukemia, non-small cell lung cancer,epithelial lung carcinoma, pancreatic carcinoma, pancreatic ductaladenocarcinoma, glioblastoma, metastatic breast cancer, melanoma, andprostate cancer. In certain embodiments, the cell proliferation disorderis osteosarcoma, promyelocytic leukemia, non-small cell lung cancer,epithelial lung carcinoma, pancreatic carcinoma, pancreatic ductaladenocarcinoma, glioblastoma, metastatic breast cancer, melanoma, orprostate cancer.

A method of inhibiting angiogenic tubule formation in a subject in needthereof includes administering to the subject an effective amount of thedisclosed compounds or compositions.

In some embodiments, the preceding methods of treating subjects or cellscan also include coadministration of an anticancer drug or a compoundthat modulates angiogenic tubule formation, particularlycoadministration of a compound that inhibits angiogenic tubuleformation. Exemplary anticancer drugs and compounds that can modulateangiogenic tubule formation are provided in the detailed description.

A method of assessing a subject for treatment with an inhibitor ofRb:Raf-1 binding interactions includes determining, in the subject or ina sample from the subject, a level of Rb, Raf-1, or Rb bound to Raf-1,wherein treatment with an inhibitor of Rb:Raf-1 binding interactions isindicated when the level of Rb, Raf-1, or Rb bound to Raf-1 is elevatedcompared to normal.

A method of identifying a subject for therapy includes the steps ofproviding a sample from the subject, determining a level of Rb, Raf-1,or Rb bound to Raf-1 in the sample; and identifying the subject fortherapy with an inhibitor of Rb:Raf-1 binding interactions when thelevel of Rb, Raf-1, or Rb bound to Raf-1 is elevated compared to normal.

A kit includes an antibody specific for Rb, Raf-1, or Rb bound to Raf-1;and instructions for determining the level of Rb, Raf-1, or Rb bound toRaf-1 in a sample using the antibody specific for Rb, Raf-1, or Rb boundto Raf-1.

In various embodiments, methods relating to cells can be conducted oncells in vitro or in vivo, particularly wherein the cell is in vivo in asubject. The subject can be, for example, a bird, a fish, or a mammal,e.g., a human.

In conclusion, the compounds, pharmaceutical compositions, and methodsof treatment described in this application are believed to be effectivefor inhibiting cellular proliferation, particularly of cells whichproliferate due to a mutation or other defect in the Rb:Raf-1 regulatorypathway. In particular, the disclosed compounds, pharmaceuticalcompositions, and methods of treatment are believed to be effective fortreating cancer and other proliferative disorders which can be inhibitedby disrupting Rb:Raf-1 binding interactions in the proliferating cells.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1A: Identification of novel Rb:Raf-1 inhibitors. Compoundsidentified in the DTP diversity set that showed the highest inhibitionof Rb:Raf-1 by ELISA. Highest scoring compounds (1) and (2) are bothbenzyl isothiourea derivatives.

FIG. 1B: Identification of novel Rb:Raf-1 inhibitors. (1) and (2) areselective for inhibiting Rb:Raf-1 and not Rb-E2F1 binding.

FIG. 1C: Identification of novel Rb:Raf-1 inhibitors. (1), (2), and theRaf-1 peptide inhibited the binding of GST Rb beads to endogenous Raf-1in U937 cell lysates.

FIG. 1D: Identification of novel Rb:Raf-1 inhibitors. Animmunoprecipitation-western blot analysis showing the disruption of theRb:Raf-1 interaction by the Raf-1 peptide-penetratin conjugate as wellas three compounds identified from the NSC library.

FIG. 2A: Compounds (1), (2), and 3a disrupted the Rb:Raf-1 interactionwith high potency. IC₅₀ values were determined using ELISA.

FIG. 2B: Inhibitors of Rb:Raf-1 interaction at 20 μM concentration donot inhibit other binding partners in ELISA.

FIG. 2C: Serum-stimulated binding of Raf-1 to Rb were inhibited byRb:Raf-1 disruptors as well as a Raf-1 peptide conjugated to penetratin.Specificity of the disruption was assessed by IP-western blots; thedrugs do not inhibit the binding of E2F1 to Rb (left panel) or MEK toRaf-1 (middle panel). Compound 3a tended to reduce the levels of cyclinD as well as its association with Rb (right panel).

FIG. 2D: Treatment with compound 3a reduced Rb phosphorylationsignificantly. Quiescent A549 cells were serum stimulated in thepresence or absence of 3a or BAY43-9002 and RB phosphorylationdetermined by western blotting.

FIG. 2E: Compound 3a does not inhibit Raf-1, cyclin D or E kinaseactivities in vitro kinase assays.

FIG. 2F: ChIP assays show that Brg1, not Raf-1 is present on quiescentA549 cdc6, cdc25A and TS promoters. Upon serum stimulation, Brg1 isdissociated from both promoters, correlating with Raf-1 binding. Serumstimulation in the presence of 3a causes the dissociation of Raf-1 andretention of Brg1 on E2F1 responsive promoters. Serum stimulation for 16hours causes dissociation of Rb, Raf-1, HP1, Brg1, and HDAC1 from thepromoters. An irrelevant antibody was used as a control forimmunoprecipitations; c-fos promoter was used as a negative control.

FIG. 3A: S-phase entry of serum stimulated U2-OS or Saos-2 cells in thepresence or absence of Rb:Raf-1 inhibitors (20 μM) was assessed by BrdUincorporation. The Rb-Raf-1 disruptors selectively arrest Rb positiveU2OS cells.

FIG. 3B: A549 cells stably transfected with a non-homologous shRNAconstruct as the control, or with two different 16 shRNA constructs thattarget Rb. Cells transfected with the Rb shRNAs show greatly reduced Rblevels.

FIG. 3C: BrdU incorporation assay showing that 20 μm of 3a does notinhibit the proliferation of A549 cells over-expressing shRNA constructsto Rb, but arrests wild-type A549 cells as well as those harboring anon-homologous, control shRNA.

FIG. 3D: BrdU incorporation assay showing the growth arrest mediated by3a in a variety of tumor cell lines harboring various mutations. 3acould effectively arrest cells with mutations in EGFR, p16, PTEN, K-Ras,and p53. In contrast cells lacking the Rb gene rendered the cellsresistant to 3a.

FIG. 3E: Over expression of E2F1 is sufficient to overcome cell cyclearrested media by 3a, while cyclin D over expression has only a partialeffect.

FIG. 3F: Compounds (1), (2) and 3a all inhibit angiogenic tubuleformation in matrigel at 20 μM.

FIG. 3G: Compound 3a inhibits adherence independent growth of severalcell lines in soft agar.

FIG. 4A: Compound 3a inhibits human tumor growth in nude mice. A549cells xenotransplanted bilaterally into the flanks of athymic nude micewere allowed to grow for 14 days until tumor volume reached 200 mm³;daily administration of 50 milligrams/kilogram (mpk) (i.p) and 150 mpk(oral) of 3a can completely inhibit tumor growth.

FIG. 4B: Compound 3a, 50 mpk administered by i.p. injection inhibitedH1650 xenograft tumor growth in nude mice.

FIG. 4C: Compound RRD-238a, a compound similar to 3a, was also able toarrest tumor growth significantly in nude mice in a similar experimentusing A549 cells. RRD238a and RRD251 were administered orally at 100mpk.

FIG. 4D, FIG. 4E: Inhibition of tumor growth was dependent on afunctional Rb protein. A549-sh6 and A549-sh8 cells (1×10⁷) wereimplanted into the flanks of nude mice. The tumors were allowed to growuntil they reached a volume of 200 mm3. Compound 3a was administered at150 mpk orally and the control group received the vehicle. Compound 3awas unable to inhibit tumor growth in tumors lacking Rb protein.

FIG. 4F: Immunohistochemical staining of tumors from mice treated with3a. Tumors were stained with Ki-67 for proliferation, pRb for cellcycle, and CD31 for angiogenesis. A dose dependent increase in apoptosisis seen by TUNEL staining.

FIG. 4G: Both doses of compound 3a inhibit the Rb:Raf-1 interaction intumor lysates without inhibiting Rb-E2F1 interaction, as seen byIP-Western blots.

FIG. 4H: Tumors maintained downregulated Rb expression till thecompletion of the experiment.

DETAILED DESCRIPTION

This application relates to compounds, pharmaceutical compositions, andmethods for modulating cell proliferation and/or Rb:Raf-1 interaction ina cell, either in vitro or in vivo. For example, disorders that can betreated with the disclosed compounds, compositions, and methods includediseases such as cancer as well as non-cancerous proliferationdisorders. Without wishing to be bound by theory, it is believed thatthe pharmaceutical activity of the disclosed compounds arises, at leastin part, to modulation of Rb:Raf-1 binding interactions by the disclosedcompound, and more particularly to disruption of Rb:Raf-1 binding.

In various embodiments, the disclosed compounds are modulators ofRb:Raf-1 binding interactions. A modulator can change the action oractivity of the molecule, enzyme, or system which it targets. Forexample, the disclosed modulators can modulate Rb:Raf 1 bindinginteractions to inhibit, disrupt, prevent, block or antagonize Rb,Raf-1, or Rb:Raf-1 binding interactions, or otherwise preventassociation or interaction between Rb and Raf-1. Thus, the disclosedcompounds can be inhibitors, disruptors, blockers, or antagonists of Rbor Raf-1 activity, or of Rb:Raf-1 binding interactions.

Thus, the compounds, pharmaceutical compositions, and methods of usedescribed in this application are believed to be effective forinhibiting cellular proliferation, particularly of cells whichproliferate due to a mutation or other defect in the Rb:Raf-1 regulatorypathway. In particular, the disclosed compounds, pharmaceuticalcompositions, and methods of use are believed to be effective fortreating cancer and other proliferative disorders which can be inhibitedby disrupting Rb:Raf-1 binding interactions in the proliferating cells.

Definitions

An aliphatic group is a straight chained, branched non-aromatichydrocarbon which is completely saturated or which contains one or moreunits of unsaturation. A cycloaliphatic group is an aliphatic group thatforms a ring. Alkyl and cycloalkyl groups are saturated aliphatic andsaturated cycloaliphatic groups, respectively. Typically, a straightchained or branched aliphatic group has from 1 to about 10 carbon atoms,typically from 1 to about 6, and preferably from 1 to about 4, and acyclic aliphatic group has from 3 to about 10 carbon atoms, typicallyfrom 3 to about 8, and preferably from 3 to about 6. An aliphatic groupis preferably a straight chained or branched alkyl group, e.g., methyl,ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, tert-butyl, pentyl,hexyl, pentyl or octyl, or a cycloalkyl group with 3 to about 8 carbonatoms. C₁₋₆ straight chained or branched alkyl or alkoxy groups or aC₃₋₈ cyclic alkyl or alkoxy group (preferably C₁₋₆ straight chained orbranched alkyl or alkoxy group) are also referred to as a “lower alkyl”or “lower alkoxy” groups; such groups substituted with —F, —Cl, —Br, or—I are “lower haloalkyl” or “lower haloalkoxy” groups; a “lowerhydroxyalkyl” is a lower alkyl substituted with —OH; and the like.

An “alkylene” group is a linking alkyl chain represented by —(CH₂)_(n)—,wherein n, the number of “backbone” atoms in the chain, is an integerfrom 1-10, typically 1-6, and preferably 1-4. An “alkenylene” group is alinking alkyl chain having one or more double bonds, wherein the numberof backbone atoms is an integer from 1-10, typically 1-6, and preferably1-4. An “alkynylene” group is a linking alkyl chain having one or moretriple bonds and optionally one or more double bonds, wherein the numberof “backbone” atoms is an integer from 1-10, typically 1-6, andpreferably 1-4.

“Heteroalkylene,” “heteroalkenylene,” and “heteroalkynylene” groups arealkylene, alkenylene, and alkynylene groups, respectively, wherein oneor more carbons are replaced with heteroatoms such as N, O, or S.

A heterocyclic group is a non-aromatic cycloaliphatic group which hasfrom 3 to about 10 ring atoms, typically from 3 to about 8, andpreferably from 3 to about 6, wherein one or more of the ring atoms is aheteroatom such as N, O, or S in the ring. Examples of heterocyclicgroups include oxazolinyl, thiazolinyl, oxazolidinyl, thiazolidinyl,tetrahydrofuranyl, tetrahyrothiophenyl, morpholino, thiomorpholino,pyrrolidinyl, piperazinyl, piperidinyl, thiazolidinyl, and the like.

The term “aryl” refers to C₆₋₁₄ carbocyclic aromatic groups such asphenyl, biphenyl, and the like. Aryl groups also include fusedpolycyclic aromatic ring systems in which a carbocyclic aromatic ring isfused to other aryl, cycloalkyl, or cycloaliphatic rings, such asnaphthyl, pyrenyl, anthracyl, 9,10-dihydroanthracyl, fluorenyl, and thelike.

The term “heteroaryl” refers to 5-14 membered aryl groups having 1 ormore O, S, or N heteroatoms. Examples of heteroaryl groups includepyridyl, pyrimidyl, pyrazinyl, triazinyl, pyranyl, pyrrolyl, imidazolyl,pyrazolyl, 1,2,3-trizaolyl, 1,2,4-triazolyl, tetrazolyl, thienyl,thiazoyl, isothiazolyl, furanyl, oxazolyl, isooxazolyl, and the like.Heteroaryl groups also include fused polycyclic aromatic ring systems inwhich a carbocyclic aromatic ring or heteroaryl ring is fused to one ormore other heteroaryl rings. Examples include quinolinyl, isoquinolinyl,quinazolinyl, napthyridyl, pyridopyrimidyl, benzothienyl,benzothiazolyl, benzoisothiazolyl, thienopyridyl, thiazolopyridyl,isothiazolopyridyl, benzofuranyl, benzooxazolyl, benzoisooxazolyl,furanopyridyl, oxazolopyridyl, isooxazolopyridyl, indolyl, isoindolyl,benzimidazolyl, benzopyrazolyl, pyrrolopyridyl, isopyrrolopyridyl,imidazopyridyl, pyrazolopyridyl, and the like. A ring recited as asubstituent herein can be bonded via any substitutable atom in the ring.

Suitable optional substituents for a substitutable atom in the precedinggroups, e.g., alkyl, cycloalkyl, aliphatic, cycloaliphatic, alkylene,alkenylene, alkynylene, heteroalkylene, heteroalkenylene,heteroalkynylene, heterocyclic, aryl, and heteroaryl groups, are thosesubstituents that do not substantially interfere with the pharmaceuticalactivity of the disclosed compounds. A “substitutable atom” is an atomthat has one or more valences or charges available to form one or morecorresponding covalent or ionic bonds with a substituent. For example, acarbon atom with one valence available (e.g., —C(—H)═) can form a singlebond to an alkyl group (e.g., —C(-alkyl)=), a carbon atom with twovalences available (e.g., —C(H₂)—) can form one or two single bonds toone or two substituents (e.g., —C(alkyl)(H)—, —C(alkyl)(Br))—,) or adouble bond to one substituent (e.g., —C(═O)—), and the like.Substitutions contemplated herein include only those substitutions thatform stable compounds.

For example, suitable optional substituents for substitutable carbonatoms include —F, —Cl, —Br, —I, —CN, —NO₂, —OR^(a), —C(O)R^(a),—OC(O)R^(a), —C(O)OR^(a), —SR^(a), —C(S)R^(a), —OC(S)R^(a), —C(S)OR^(a),—C(O)SR^(a), —C(S)SR^(a), —S(O)R^(a), —SO₂R^(a), —SO₃R^(a), —OSO₂R^(a),—OSO₃R^(a), —PO₂R^(a)R^(b), —OPO₂R^(a)R^(b), —PO₃R^(a)R^(b),—OPO₃R^(a)R^(b), —N(R^(a)R^(b)), —C(O)N(R^(a)R^(b)),—C(O)NR^(a)NR^(b)SO₂R^(c), —C(O)NR^(a)SO₂R^(c), —C(O)NR^(a)CN,—SO₂N(R^(a)R^(b)), —SO₂N(R^(a)R^(b)), —NR^(c)C(O)R^(a),—NR^(c)C(O)OR^(a), NR^(c)C(O)N(R^(a)R^(b)), —C(NR^(c))—N(R^(a)R^(b)),—NR^(d)—C(NR^(c))—N(R^(a)R^(b)), —NR^(a)N(R^(a)R^(b)),—CR^(c)═CR^(a)R^(b), —C≡CR^(a), ═O, ═S, ═CR^(a)R^(b), ═NR^(a), ═NOR^(a),═NNR^(a), optionally substituted alkyl, optionally substitutedcycloalkyl, optionally substituted aliphatic, optionally substitutedcycloaliphatic, optionally substituted heterocyclic, optionallysubstituted benzyl, optionally substituted aryl, and optionallysubstituted heteroaryl, wherein R^(a)-R^(d) are each independently —H oran optionally substituted aliphatic, optionally substitutedcycloaliphatic, optionally substituted heterocyclic, optionallysubstituted benzyl, optionally substituted aryl, or optionallysubstituted heteroaryl, or, —N(R^(a)R^(b)), taken together, is anoptionally substituted heterocyclic group.

Suitable substituents for nitrogen atoms having two covalent bonds toother atoms include, for example, optionally substituted alkyl,optionally substituted cycloalkyl, optionally substituted aliphatic,optionally substituted cycloaliphatic, optionally substitutedheterocyclic, optionally substituted benzyl, optionally substitutedaryl, optionally substituted heteroaryl, —CN, —NO₂, —OR^(a), —C(O)R^(a),—OC(O)R^(a), —C(O)OR^(a), —SR^(a), —S(O)R^(a), —SO₂R^(a), —SO₃R^(a),—N(R^(a)R^(b)), —C(O)N(R^(a)R^(b)), —C(O)NR^(a)NR^(b)SO₂R^(c),—C(O)NR^(a)SO₂R^(c), —C(O)NR^(a)CN, —SO₂N(R^(a)R^(b)),—SO₂N(R^(a)R^(b)), —NR^(c)C(O)R^(a), —NR^(c)C(O)OR^(a),—NR^(c)C(O)N(R^(a)R^(b)), and the like.

A nitrogen-containing group, for example, a heteroaryl or non-aromaticheterocycle, can be substituted with oxygen to form an N-oxide, e.g., asin a pyridyl N-oxide, piperidyl N-oxide, and the like. For example, invarious embodiments, a ring nitrogen atom in a nitrogen-containingheterocyclic or heteroaryl group can be substituted to form an N-oxide.

Suitable substituents for nitrogen atoms having three covalent bonds toother atoms include —OH, alkyl, and alkoxy (preferably C₁₋₆ alkyl andalkoxy). Substituted ring nitrogen atoms that have three covalent bondsto other ring atoms are positively charged, which is balanced bycounteranions corresponding to those found in pharmaceuticallyacceptable salts, such as chloride, bromide, fluoride, iodide, formate,acetate and the like. Examples of other suitable counteranions areprovided in the section below directed to suitable pharmacologicallyacceptable salts.

It will also be understood that certain disclosed compounds can beobtained as different stereoisomers (e.g., diastereomers andenantiomers) and that the invention includes all isomeric forms andracemic mixtures of the disclosed compounds and methods of treating asubject with both pure isomers and mixtures thereof, including racemicmixtures. Stereoisomers can be separated and isolated using any suitablemethod, such as chromatography.

Also included in the present invention are pharmaceutically acceptablesalts of the disclosed compounds. These disclosed compounds can have oneor more sufficiently acidic protons that can react with a suitableorganic or inorganic base to form a base addition salt. When it isstated that a compound has a hydrogen atom bonded to an oxygen,nitrogen, or sulfur atom, it is contemplated that the compound alsoincludes salts thereof where this hydrogen atom has been reacted with asuitable organic or inorganic base to form a base addition salt. Baseaddition salts include those derived from inorganic bases, such asammonium or alkali or alkaline earth metal hydroxides, carbonates,bicarbonates, and the like, and organic bases such as alkoxides, alkylamides, alkyl and aryl amines, and the like. Such bases useful inpreparing the salts of this invention thus include sodium hydroxide,potassium hydroxide, ammonium hydroxide, potassium carbonate, and thelike.

For example, pharmaceutically acceptable salts of the disclosedcompounds can include those formed by the reaction of the disclosedcompounds with one equivalent of a suitable base to form a monovalentsalt (i.e., the compound has single negative charge that is balanced bya pharmaceutically acceptable counter cation, e.g., a monovalent cation)or with two equivalents of a suitable base to form a divalent salt(e.g., the compound has a two-electron negative charge that is balancedby two pharmaceutically acceptable counter cations, e.g., twopharmaceutically acceptable monovalent cations or a singlepharmaceutically acceptable divalent cation). “Pharmaceuticallyacceptable” means that the cation is suitable for administration to asubject. Examples include Lie, Na⁺, K⁺, Mg²⁺, Ca²⁺ and NR₄ ⁺, whereineach R is independently hydrogen, an optionally substituted aliphaticgroup (e.g., a hydroxyalkyl group, aminoalkyl group or ammoniumalkylgroup) or optionally substituted aryl group, or two R groups, takentogether, form an optionally substituted non-aromatic heterocyclic ringoptionally fused to an aromatic ring. Generally, the pharmaceuticallyacceptable cation is Lie, Na⁺, K⁺, NH₃(C₂H₅OH)⁺ or N(CH₃)₃(C₂H₅OH)⁺.

Pharmaceutically acceptable salts of the disclosed compounds with asufficiently basic group, such as an amine, can be formed by reaction ofthe disclosed compounds with an organic or inorganic acid to form anacid addition salt. Acids commonly employed to form acid addition saltsfrom compounds with basic groups can include inorganic acids such ashydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid,phosphoric acid, and the like, and organic acids such asp-toluenesulfonic acid, methanesulfonic acid, oxalic acid,p-bromophenyl-sulfonic acid, carbonic acid, succinic acid, citric acid,benzoic acid, acetic acid, and the like. Examples of such salts includethe sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate,monohydrogenphosphate, dihydrogenphosphate, metaphosphate,pyrophosphate, chloride, bromide, iodide, acetate, propionate,decanoate, caprylate, acrylate, formate, isobutyrate, caproate,heptanoate, propiolate, oxalate, malonate, succinate, suberate,sebacate, fumarate, maleate, butyne-1,4-dioate, hexyne-1,6-dioate,benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate,hydroxybenzoate, methoxybenzoate, phthalate, sulfonate, xylenesulfonate,phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate,gamma-hydroxybutyrate, glycolate, tartrate, methanesulfonate,propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate,mandelate, and the like. In certain embodiments, the disclosed compoundforms a pharmaceutically acceptable salt with HCl, HF, HBr, HI,trifluoracetic acid, or sulfuric acid. In particular embodiments, thedisclosed compound forms a pharmaceutically acceptable salt withsulfuric acid. Also included are pharmaceutically acceptable solvates.As used herein, the term “solvate” means a compound of the presentinvention or a salt thereof, that further includes a stoichiometric ornon-stoichiometric amount of solvent, e.g., water or organic solvent,bound by non-covalent intermolecular forces.

Preparation Methods

Synthetic chemistry functional group transformations useful insynthesizing the disclosed compounds are known in the art and include,for example, those described in R. Larock, Comprehensive OrganicTransformations, VCH Publishers (1989); L. Fieser and M. Fieser, Fieserand Fieser's Reagents for Organic Synthesis, John Wiley and Sons (1994);and L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis,John Wiley and Sons (1995). The entire teachings of these documents areincorporated herein by reference. For example, suitable techniques forconverting the —OH group in the third reagent represented by formula IVto the amine or leaving group represented by variable W in the firstreagent represented by formula Ia are well known. In particular, the —OHin structural formula IV can be converted to —Cl, for example, using achlorinating reagent such as thionyl chloride or N-chlorosuccinimide incombination with ultraviolet irradiation. Also, typical reagents andconditions for reducing carbonyls to alcohols (e.g., carbonyl Vb toalcohol IV) are well-known, for example, lithium aluminum hydride,sodium borohydride, lithium hydride, sodium hydride, potassium hydride,hydrogen in the presence of a catalyst, e.g., Pd or Pt on carbon,electrochemical methods, and the like. Further, typical reagents andconditions for preparing Grignard or organoalkali derivatives of R^(y)′to convert carbonyl Vb to alcohol IV such as R^(y)′MgCl, R^(y)′MgBr,R^(y)′MgI, R^(y)′Li, R^(y)′Na, or R^(y)′K can be readily prepared fromthe corresponding R^(y)′-halide, e.g., when R^(y)′ is C₁₋₆ alkyl, C₁₋₆aralkyl, or aryl.

As used herein, “suitable protecting groups” and strategies forprotecting and deprotecting functional groups using protecting groupsuseful in synthesizing the disclosed compounds are known in the art andinclude, for example, those described in T. W. Greene and P. G. M. Wuts,Protective Groups in Organic Synthesis, 2nd Ed., John Wiley and Sons(1991), ), the entire teachings of which are incorporated herein byreference. For example, suitable hydroxyl protecting groups include, butare not limited to substituted methyl ethers (e.g., methoxymethyl,benzyloxymethyl) substituted ethyl ethers (e.g., ethoxymethyl,ethoxyethyl)benzyl ethers (benzyl, nitrobenzyl, halobenzyl) silyl ethers(e.g., trimethylsilyl), esters, and the like. Examples of suitable amineprotecting groups include benzyloxycarbonyl, tert-butoxycarbonyl,tert-butyl, benzyl and fluorenylmethyloxy-carbonyl (Fmoc). Examples ofsuitable thiol protecting groups include benzyl, tert-butyl, acetyl,methoxymethyl and the like.

The reactions described herein may be conducted in any suitable solventfor the reagents and products in a particular reaction. Suitablesolvents are those that facilitate the intended reaction but do notreact with the reagents or the products of the reaction. Suitablesolvents can include, for example: ethereal solvents such as diethylether or tetrahydrofuran; ketone solvents such as acetone or methylethyl ketone; halogenated solvents such as dichloromethane, chloroform,carbon tetrachloride, or trichloroethane; aromatic solvents such asbenzene, toluene, xylene, or pyridine; polar aprotic organic solventssuch as acetonitrile, dimethyl sulfoxide, dimethyl formamide, N-methylpyrrolidone, hexamethyl phosphoramide, nitromethane, nitrobenzene, orthe like; polar protic solvents such as methanol, ethanol, propanol,butanol, ethylene glycol, tetraethylene glycol, or the like; nonpolarhydrocarbons such as pentane, hexane, cyclohexane, cyclopentane,heptane, octane, or the like; basic amine solvents such as pyridine,triethyleamine, or the like; and other solvents known to the art.

Reactions or reagents which are water sensitive may be handled underanhydrous conditions. Reactions or reagents which are oxygen sensitivemay be handled under an inert atmosphere, such as nitrogen, helium,neon, argon, and the like. Reactions or reagents which are lightsensitive may be handled in the dark or with suitably filteredillumination.

Reactions or reagents which are temperature-sensitive, e.g., reagentsthat are sensitive to high temperature or reactions which are exothermicmay be conducted under temperature controlled conditions. For example,reactions that are strongly exothermic may be conducted while beingcooled to a reduced temperature.

Reactions that are not strongly exothermic may be conducted at highertemperatures to facilitate the intended reaction, for example, byheating to the reflux temperature of the reaction solvent. Reactions canalso be conducted under microwave irradiation conditions. For example,in various embodiments of the method, the first and second reagents arereacted together under microwave irradiation.

Reactions may also be conducted at atmospheric pressure, reducedpressure compared to atmospheric, or elevated pressure compared toatmospheric pressure. For example, a reduction reaction may be conductedin the presence of an elevated pressure of hydrogen gas in combinationwith a hydrogenation catalyst.

Reactions may be conducted at stoichiometric ratios of reagents, orwhere one or more reagents are in excess. For example, in forming thecompound, e.g., represented by formula Ia, using the first reagent,e.g., represented by IIa and the second reagent, e.g., represented byformula IIIa, the first reagent may be used in a molar ratio to thesecond reagent of about 20:1, 10:1, 5:1, 2.5:1, 2:1, 1.5:1, 1.3:1,1.2:1, 1.1:1, 1:1, 0.91:1, 0.83:1, 0.77:1, 0.67:1, 0.5:1, 0.4:1, 0.2:1,0.1:1 or 0.5:1. Typically, the first reagent may be used in a molarratio to the second reagent of about 5:1, 2.5:1, 2:1, 1.5:1, 1.3:11.2:1, 1.1:1, 1:1, 0.91:1, 0.83:1, 0.77:1, 0.67:1, 0.5:1, 0.4:1. Incertain embodiments, the first reagent may be used in a molar ratio tothe second reagent of about 1.5:1, 1.3:1, 1.2:1, 1.1:1, 1:1, 0.91:1,0.83:1, 0.77:1, or 0.67:1. Preferably, first reagent may be used in amolar ratio to the second reagent of between about 1.1:1 and 0.9:1,typically about 1:1. The same ratios may be used for other reagents inthe reaction. For example, when the first reagent and the second reagentare reacted together in the presence of a pyridinium salt (e.g.,1-alkyl-2-halopyridinium salt or an optionally substituted1-aryl-2-halopyridinium salt), the first reagent may be used in a molarratio to the pyridinium salt independently selected from the precedingranges of ratios between the first reagent and the second reagent.Likewise, when the third reagent is prepared by reacting a fourthreagent represented by structural formula Va with a Grignard ororganoalkali (e.g., R^(y)′MgCl, R^(y)′MgBr, R^(y)′MgI, R^(y)′Li,R^(y)′Na, or R^(y)′K, wherein R^(y)′ is C₁₋₆ alkyl, C₁₋₆ aralkyl, oraryl), the fourth reagent may be used in a molar ratio to the Grignardor organoalkali independently selected from the preceding ranges ofratios between the first reagent and the second reagent. Similarly, whenthe fourth reagent is prepared by reducing the fifth reagent, the ratiobetween the reducing agent and the fifth reagent can be independentlyselected from the preceding ranges of ratios between the first reagentand the second reagent.

Assay Methods

The disclosed compounds can be assayed for binding and biologicalactivity by any means described herein or known to the art. For example,the disclosed compounds can be screened for binding activity in an ELISAassay (see Methods, Example 1), the IC₅₀ values of the disclosedcompounds can be determined by in vitro binding assays (see Methods,Example 4), the binding selectivity of the disclosed compounds can bemeasured in competitive ELISA assays (see Example 5 and 8), and theability of the disclosed compounds to disrupt Rb:Raf-1 in vitro (seeExample 6) or in vivo (see Example 7) can be assayed.

Further, the disclosed compounds can be tested for their ability to killor inhibit the growth of tumor cells or angiogenic tubules. Suitableassays include, for example, (a) tumor cell in anchorage/independentgrowth (soft agar assays, see Methods and Example 19) (b) tumor cell inanchorage-dependent growth(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT),trypan blue and DNA synthesis assays) (c) tumor cell survival (TUNEL,PARP cleavage, caspace activation and other apoptosis assays, seeMethods and Example 20) (d) tumor cell invasion and metastasis (seemethods) (e) endothelial cell migration, invasion and angiogenesis (seeMethods and Example 18) (f) tumor cell proliferation inhibition assays(see Examples 9-16) (g) anti-tumor activity assays in animal models (seeExample 20), and other such assays known to the art.

Certain assays can be used to assess a subject for treatment with aninhibitor of Rb:Raf-1 binding interactions or to identify a subject fortherapy. The level of Rb, Raf-1, or Rb bound to Raf-1 can be determinedin the subject or in a sample from the subject, e.g., a subject with acell proliferation disorder. treatment with the disclosed compounds isindicated when the level of Rb, Raf-1, or Rb bound to Raf-1 is elevatedcompared to normal. “Elevated compared to normal” means that the levelsare higher than in a reference sample of cells of the same type that arehealthy. For example, the level of Rb, Raf-1, or Rb bound to Raf-1 incells from a non-small cell lung cancer tumor can be compared to thelevel of Rb, Raf-1, or Rb bound to Raf-1 in normal, noncancerous cells.For example, Enzyme Linked ImmunoSorbent Assay (ELISA) can be used incombination with antibodies to Rb, Raf-1, or Rb bound to Raf-1 (seeMethods, In vitro library screening assays and Example 5). The assay canbe embodied in a kit. For example, a kit includes a reagent orindicator, such as an antibody, that is specific for Rb, Raf-1, or Rbbound to Raf-1. The kit can also include instructions for determiningthe level of Rb, Raf-1, or Rb bound to Raf-1 in a sample using thereagent or indicator, such as an antibody, that is specific for Rb,Raf-1, or Rb bound to Raf-1.

Utility

In various embodiments, methods relating to cells can be conducted oncells in vitro or in vivo, particularly wherein the cell is in vivo,i.e., the cell is located in a subject. A “subject” can be any animalwith a proliferative disorder, for example, mammals, birds, reptiles, orfish. Preferably, the animal is a mammal. More preferably, the mammal isselected from the group consisting of dogs, cats, sheep, goats, cattle,horses, pigs, mice, non-human primates, and humans. Most preferably, themammal is a human.

Disease Indications

As used herein, a “cell proliferation disorder” includes cancer andnon-cancerous cell proliferation disorders. In some embodiments, thecell proliferation disorder is angiogenesis or the cell proliferationdisorder is mediated by angiogenesis.

As used herein, a “cancer” includes, for example, fibrosarcoma,myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma,angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer,breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma,basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceousgland carcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testiculartumor, lung carcinoma, small cell lung carcinoma, non-small cell lungcarcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma,melanoma, neuroblastoma, retinoblastoma, acute lymphocytic leukemia,lymphocytic leukemia, large granular lymphocytic leukemia, acutemyelocytic leukemia, chronic leukemia, polycythemia vera, Hodgkin'slymphoma, non-Hodgkin's lymphoma, multiple myeloma, Waldenstrobm'smacroglobulinemia, heavy chain disease, lymphoblastic leukemia, T-cellleukemia, T-lymphocytic leukemia, T-lymphoblastic leukemia, B cellleukemia, B-lymphocytic leukemia, mixed cell leukemias, myeloidleukemias, myelocytic leukemia, myelogenous leukemia, neutrophilicleukemia, eosinophilic leukemia, monocytic leukemia, myelomonocyticleukemia, Naegeli-type myeloid leukemia, nonlymphocytic leukemia,osteosarcoma, promyelocytic leukemia, non-small cell lung cancer,epithelial lung carcinoma, pancreatic carcinoma, pancreatic ductaladenocarcinoma, glioblastoma, metastatic breast cancer, melanoma, orprostate cancer.

In various embodiments, the cancer includes cells that have a mutationor defect in the Rb:Raf-1 pathway. In certain embodiments, the cancer isosteosarcoma, promyelocytic leukemia, non-small cell lung cancer,epithelial lung carcinoma, pancreatic carcinoma, pancreatic ductaladenocarcinoma, glioblastoma, metastatic breast cancer, melanoma, orprostate cancer.

In various embodiments, the non-cancerous cell proliferation disorderincludes cells that have a mutation or defect in the Rb:Raf-1 pathway. Anon-cancerous cell proliferation disorder can include, for example,smooth muscle cell proliferation, systemic sclerosis, cirrhosis of theliver, adult respiratory distress syndrome, idiopathic cardiomyopathy,lupus erythematosus, retinopathy, cardiac hyperplasia, benign prostatichyperplasia, ovarian cysts, pulmonary fibrosis, endometriosis,fibromatosis, harmatomas, lymphangiomatosis, sarcoidosis, desmoidtumors, intimal smooth muscle cell hyperplasia, restenosis, vascularocclusion, hyperplasia in the bile duct, hyperplasia in the bronchialairways, hyperplasia in the kidneys of patients with renal interstitialfibrosis, psoriasis, Reiter's syndrome, pityriasis rubra pilaris, ahyperproliferative disorder of keratinization, or scleroderma.

Pharmaceutical Compositions and Formulations

Also included are pharmaceutical compositions comprising the disclosedcompounds. A “pharmaceutical composition” comprises a disclosedcompound, typically in conjunction with an acceptable pharmaceuticalcarrier as part of a pharmaceutical composition for administration to asubject.

Suitable formulations for administration include, for example, injectioncompositions, infusion compositions, topical administration solutions,emulsions, capsules, creams, ointments, tablets, pills, lozenges,suppositories, depot preparations, implanted reservoirs, intravaginalrings, coatings on implantable medical devices (e.g., a stent),impregnation in implantable medical devices, and the like. Suitablepharmaceutical carriers may contain inert ingredients which do notinteract with the compound. Standard pharmaceutical formulationtechniques can be employed, such as those described in Remington'sPharmaceutical Sciences, Mack Publishing Company, Easton, Pa. Suitablepharmaceutical carriers for parenteral administration include, forexample, sterile water, physiological saline, bacteriostatic saline(saline containing about 0.9% mg/mL benzyl alcohol), phosphate-bufferedsaline, Hank's solution, Ringer's-lactate and the like. Methods forencapsulating compositions (such as in a coating of hard gelatin orcyclodextrasn) are known in the art (Baker, et al., “Controlled Releaseof Biological Active Agents”, John Wiley and Sons, 1986).

For example, a sterile injectable composition such as a sterileinjectable aqueous or oleaginous suspension, can be formulated accordingto techniques known in the art using suitable dispersing or wettingagents (such as, for example, Tween 80) and suspending agents. Thesterile injectable preparation can also be a sterile injectable solutionor suspension in a non-toxic parenterally acceptable diluent or solvent,for example, as a solution in 1,3-butanediol. Other examples ofacceptable vehicles and solvents include mannitol, water, Ringer'ssolution and isotonic sodium chloride solution. In addition, sterile,fixed oils are conventionally employed as a solvent or suspending medium(e.g., synthetic mono- or diglycerides). Fatty acids, such as oleic acidand its glyceride derivatives can be useful in the preparation ofinjectables, as well as natural pharmaceutically-acceptable oils, suchas olive oil or castor oil, for example in their polyoxyethylatedversions. Oil solutions or suspensions can also contain a long-chainalcohol diluent or dispersant, or carboxymethyl cellulose or similardispersing agents.

A composition for oral administration, for example, can be any orallyacceptable dosage form including, but not limited to, capsules, tablets,emulsions and aqueous suspensions, dispersions and solutions. In thecase of tablets for oral use, carriers which are commonly used includelactose and corn starch. Lubricating agents, such as magnesium stearate,are also typically added. For oral administration in a capsule form,useful diluents include lactose and dried corn starch. When aqueoussuspensions or emulsions are administered orally, the active ingredientcan be suspended or dissolved in an oily phase combined with emulsifyingor suspending agents. If desired, certain sweetening, flavoring, orcoloring agents can be added. A nasal aerosol or inhalation compositioncan be prepared according to techniques well-known in the art ofpharmaceutical formulation and can be prepared as solutions in saline,employing benzyl alcohol or other suitable preservatives, absorptionpromoters to enhance bioavailability, fluorocarbons, and/or othersolubilizing or dispersing agents known in the art.

As used herein, the term “pharmaceutically acceptable” means that thematerials (e.g., compositions, carriers, diluents, reagents, salts, andthe like) are capable of administration to or upon a mammal with aminimum of undesirable physiological effects such as nausea, dizzinessor gastric upset.

Mode of Administration

Formulation of the compound to be administered will vary according tothe route of administration selected, e.g., parenteral, oral, buccal,epicutaneous, inhalational, opthalamic, intraear, intranasal,intravenous, intraarterial, intramuscular, intracardiac, subcutaneous,intraosseous, intracutaneous, intradermal, intraperitoneal, topically,transdermal, transmucosal, intraarticular, intrasynovial, intrasternal,intralesional, intracranial inhalational, insufflation, pulmonary,epidural, intratumoral, intrathecal, vaginal, rectal, or intravitrealadministration.

An “effective amount” to be administered is the quantity of compound inwhich a beneficial outcome is achieved when the compound is administeredto a subject or alternatively, the quantity of compound that possess adesired activity in vivo or in vitro. In the case of cell proliferationdisorders, a beneficial clinical outcome includes reduction in theextent or severity of the symptoms associated with the disease ordisorder and/or an increase in the longevity and/or quality of life ofthe subject compared with the absence of the treatment. The preciseamount of compound administered to a subject will depend on the type andseverity of the disease or condition and on the characteristics of thesubject, such as general health, age, sex, body weight and tolerance todrugs. It will also depend on the degree, severity and type of disorder.The skilled artisan will be able to determine appropriate dosagesdepending on these and other factors. The interrelationship of dosagesfor animals and humans (based on milligrams per meter squared of bodysurface) is described, for example, in Freireich et al., (1966) CancerChemother Rep 50: 219. Body surface area may be approximately determinedfrom height and weight of the patient. See, e.g., Scientific Tables,Geigy Pharmaceuticals, Ardley, N.Y., 1970, 537. An effective amount ofthe disclosed compounds can range from about 0.001 mg/kg to about 1000mg/kg, more preferably 0.01 mg/kg to about 500 mg/kg, more preferably 1mg/kg to about 200 mg/kg. Effective doses will also vary, as recognizedby those skilled in the art, depending on the diseases treated, route ofadministration, excipient usage, and the possibility of co-usage withother therapeutic treatments such as use of other agents.

The disclosed compounds can be co-administered with anti-cancer agentsor chemotherapeutic agents such as alkylating agents, antimetabolites,natural products, hormones, metal coordination compounds, or otheranticancer drugs. Examples of alkylating agents include nitrogenmustards (e.g., cyclophosphamide), ethylenimine and methylmelamines(e.g., hexamethlymelamine, thiotepa), alkyl sulfonates (e.g., busulfan),nitrosoureas (e.g., streptozocin), or triazenes (decarbazine, etc.).Examples of antimetabolites include folic acid analogs (e.g.,methotrexate), pyrimidine analogs (e.g., fluorouracil), purine analogs(e.g., mercaptopurine). Examples of natural products include vincaalkaloids (e.g., vincristine), epipodophyllotoxins (e.g., etoposide),antibiotics (e.g., doxorubicin,), enzymes (e.g., L-asparaginase), orbiological response modifiers (e.g., interferon alpha). Examples ofhormones and antagonists include adrenocorticosteroids (e.g.,prednisone), progestins (e.g., hydroxyprogesterone), estrogens (e.g.,diethlystilbestrol), antiestrogen (e.g., tamoxifen), androgens (e.g.,testosterone), antiandrogen (e.g., flutamide), and gonadotropinreleasing hormone analog (e.g., leuprolide). Other agents that can beused in the methods and with the compositions of the invention for thetreatment or prevention of cancer include platinum coordinationcomplexes (e.g., cisplatin, carboblatin), anthracenedione (e.g.,mitoxantrone), substituted urea (e.g., hydroxyurea), methyl hydrazinederivative (e.g., procarbazine), or adrenocortical suppressants (e.g.,mitotane).

In various embodiments compounds can be coadministered with compoundsthat can inhibit angiogenesis or inhibit angiogenic tubule formationinclude, for example, matrix metalloproteinase inhibitors (dalteparin,suramin), endothelial cell inhibitors (e.g., thalidomide, squalamine,2-methoxyestradiol), inhibitors of angiogenesis activation (e.g.,avastatin, endostatin), celecoxib and the like.

Methods

Chemistry. ¹H NMR spectra were recorded using a Mercury 400 NMRspectrometer (Varian, Palo Alto, Calif.). ¹³C NMR spectra were recordedusing Distortionless Enhancement by Polarization Transfer. Both ¹H and¹³C spectra were recorded using CDCl₃ or d₆-DMSO (dimethyl sulfoxide) asinternal standard. Atmospheric pressure ionization (API) andelectrospray (ES) mass spectra and accurate mass determinations wererecorded using a time of flight (TOF) mass spectrometer (anAgilent/Hewlett Packard, Santa Clara, Calif.). High Performance LiquidChromatography (HPLC) analysis was performed using a HPLC systemequipped with a PU-2089 Plus quaternary gradient pump and a UV-2075 PlusUV-VIS detector (JASCO, Easton, Md.). Infra red spectra were recordedusing a FTIR-4100 spectrometer (JASCO). Melting points were determinedusing a MEL-TEMP Electrothermal melting point apparatus and wereuncorrected. Column chromatography was conducted using silica gel 63-200mesh (Merck & Co., Whitehouse Station, N.J.). Silica thin layerchromatography (TLC) was conducted on pre-coated aluminum sheets (60F₂₅₄, Merck & Co.).

Cell culture and transfection. The human promyelocytic leukemia cellline U937 was cultured in RPMI (Mediatech, Hernden, Va.) containing 10%fetal bovine serum (FBS; Mediatech). U2-OS, Saos-2, MCF7, PANC1 andMDA-MB-231 cell lines were cultured in Dulbecco modified Eagle Medium(DMEM; Mediatech) containing 10% FBS. A549 cells and A549 shRNA Rb celllines were maintained in Ham F-12K supplemented with 10% FBS. ShRNAcells lines were maintained in media containing 0.5 μg/mL puromycin.H1650, PC-9 and Aspc1 cell line were cultured in RPMI (Gibco/Invitrogen,Carlsbad, Calif.) containing 10% FBS. PANC1 and CAPAN2 pancreatic celllines and the A375 Melanoma cell line was grown in DMEM supplementedwith 10% FBS. Human aortic endothelial cells (HAECs, Clonetics, SanDiego, Calif.) were cultured in endothelial growth medium, supplementedwith 5% FBS, according to the manufacturer's instructions. U251MG andU87MG glioma cell lines were maintained in DMEM supplemented withnon-essential amino acids, 50 mM β-mercaptoethanol, and 10% FBS. ShRNAcell lines were made by stably transfecting A549 cells with twodifferent shRNA constructs that specifically target Rb obtained from alibrary. The adenovirus (Ad) constructs Ad-green fluorescent protein(GFP) and Ad-E2F1 were obtained from W. D. Cress. Ad-cyclin D wasprovided by I. Cozar-Castellano.

In vitro library screening assays. Enzyme Linked ImmunoSorbent Assay(ELISA) 96-well plates were coated with 1 μg/mL of a glutathioneS-transferase (GST) Raf-1 (1-149aa) overnight at 4° C. Subsequently theplates were blocked and GST Rb at 20 μg/mL was rotated at roomtemperature (RT) for 30 minutes in the presence or absence of thecompounds at 20 micromolar (μM). GST-Rb+/−compounds were then added tothe plate and incubated for 90 minutes (min) at 37° C. The amount of Rbbound to Raf-1 was detected by Rb polyclonal antibody (Santa CruzBiotechnology, Santa Cruz, Calif.) 1:1000 incubated for 60 min at 37° C.Donkey-anti-rabbit-IgG-HRP (1:10,000) was added to the plate andincubated at 37° C. for 60 minutes. The color was developed withorthophenylenediamine (Sigma, St. Louis, Mo.) and the reaction wasterminated with 3 molar (M) H₂SO₄. Absorbance was read at 490 nanometers(nm). To determine disruption of Rb to E2F1, Phb, or HDAC1 the aboveprotocol was used with the exception of coating GST Rb on the ELISAplate and adding the drugs in the presence or absence of GST E2F1, Phb,or HDAC1. E2F1 monoclonal antibody (1:2000) was used to detect theamount of Rb bound to E2F1. Prohibitin monoclonal antibody was used at1:1000 to detect the amount of Rb bound to Prohibitin. For disruption ofMEK-Raf-1 binding ELISAs, Raf-1 1 microgram/milliliter (μg/mL) wascoated on the plate and GST-MEK (20 μg/mL) was incubated+/−the compoundsfor 30 minutes at room temperature. Mek1 polyclonal antibody was used at1:1000 to detect the binding of Raf-1 to Mek1. The IC₅₀ concentrationsfor the Rb:Raf-1 inhibitors were determined by plotting with Origin 7.5software (Origin, Northampton, Mass.).

In vitro binding assays. Glutathione S-transferase (GST) fusion of Rb,Raf-1, E2F1, and MEK1 have been previously described (Dasgupta P, Sun J,Wang S, et al. Mol Cell Biol 2004; 24(21):9527-9541). First, 200micrograms (μg) of U937 asynchronous lysates were pre-incubated with 10μM of the indicated drugs or 1 μM of the Raf-1 peptide for 30 minutes at4° C. Next, 200 μg of the U937 lysates were incubated with glutathionebeads carrying an equal amount of the GST fusion proteins in 200 μl ofprotein binding buffer (20 mM Tris [pH 7.5], 50 mM KCL, 0.5 mM EDTA, 1mM dithiothreitol, 0.5% NP-40, 3 mg of bovine serum albumin/mL) at 4° C.for 2 h. (Wang S, Ghosh R, Chellappan S. Mol Cell Biol 1998;18(12):7487-7498).

Matrigel Assays. Matrigel (Collaborative Biomedical Products) was usedto promote the differentiation of HAECs into capillary tube-likestructures (Dasgupta P, Sun J, Wang S, et al. Mol Cell Biol 2004;24(21):9527-9541). A total of 100 μl of thawed Matrigel was added to96-well tissue culture plates, followed by incubation at 37° C. for 60minutes to allow polymerization. Subsequently, 1×10⁴ HAECs were seededon the gels in EGM medium supplemented with 5% FBS in the presence orabsence of 20 μM concentrations of the indicated compounds, followed byincubation for 24 hours at 37° C. Capillary tube formation assessed byusing a Leica DMIL phase contrast microscope.

Lysate preparation, immunoprecipitation, and Western blotting. Lysatesfrom cells treated with different agents were prepared by NP-40 lysis asdescribed earlier (Wang 1998). Tumor lysates were prepared with T-Pertissue lysis buffer (Pierce) and a Fischer PowerGen 125 douncehomogenizer. Physical interaction between proteins in vivo was analyzedby immunoprecipitation—Western blot analyses with 200 μg of lysate with1 μg of the indicated antibody as previously described (Wang 1998).Polyclonal E2F1 and Cyclin D were obtained from Santa CruzBiotechnology. Monoclonal Rb and Raf-1 were supplied by BD Transductionlaboratories (San Jose, Calif.). Polyclonal antibodies to phospho-Rb(807,811) phospho-MEK1/2, MEK1/2, phospho-Erk1/2 and ERK1/2 weresupplied by Cell Signaling (Danvers, Mass.).

Chromatin Immunoprecipitation (ChIP) assay. A549 cells were renderedquiescent by serum starvation and re-stimulated with serum for 2 h or 16h in the presence or absence of RRD 251 at 20 μM. Cells werecross-linked with 1% formaldehyde for 10 minutes at room temperature.Subsequently, the cells were harvested and lysates were prepared.Immunoprecipitations were analyzed for the presence of E2F1, Rb, Raf-1,Brg1, HP1, and HDAC1 by PCR as previously described (Dasgupta 2004).Rabbit anti-mouse secondary antibody was used as the control for allreactions. The sequences of the PCR primers used in the PCRs were asfollows: Cdc6 promoter (forward primer), 5′-GGCCTCACAG CGACTCTAAGA-3′;and Cdc6 promoter (reverse primer), 5′-CTCGGACTCACCACAAGC-3′. TSpromoter (forward primer), and 5′-GAC GGA GGC AGG CCA AGT G-3′ TSpromoter (reverse primer). The cdc25A and c-fos primers are described in(Dasgupta, 2004).

In vitro kinase assay. The kinase reaction for Raf-1 was carried outwith 100 nanograms (ng) of Raf-1 (Upstate Signaling, Charlottesville,Va.), 0.5 μg of full-length Rb protein (QED Bioscience, San Diego,Calif.) as the substrate, 10 μM ATP, 10 μCi of [γ-³²P] ATP in the kinaseassay buffer in the presence or absence of the drugs at 30° C. for 30minutes. Cyclin D and E kinase assays are described in (Dasgupta 2004).

Proliferation assays. Bromodeoxyuridine (BrdU) labeling kits wereobtained from Roche Biochemicals (Indianapolis, Ind.). Cells were platedin poly-D-lysine coated chamber slides at a density of 10,000 cells perwell and rendered quiescent by serum starvation for 24 hours. Cells werethen re-stimulated with serum in the presence or absence of theindicated drugs for 18 h. S-phase cells were visualized by microscopyand quantitated by counting 3 fields of 100 in quadruplicate.

Soft Agar assay. Soft agar assays were done in triplicate in 12-wellplates (Corning, Corning N.Y.). First, the bottom layer of agar (0.6%)was allowed to solidify at room temperature. Next the top layer of agarwas (0.3%) was mixed with 5,000 cells per well and the indicated drug.The drugs were added twice weekly in complete media to the agar wells.Colonies were quantified by staining with MTT(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) 1 mg/mLfor 1 hour at 37° C.

Animal Studies. Nude mice (Charles River, Wilminton, Mass., USA) weremaintained in accordance with Institutional Animal Care and UseCommittee (IAUCUC) procedures and guidelines. A549 cells were harvestedand resuspended in PBS, and then injected s.c. into the right and leftflanks (10×10⁶ cells per flank) of 8-week old female nude mice asreported previously (Sun 99). When tumors reached about 100-200 mm³,animals were dosed intraperitoneally i.p. or orally by gavage with 0.1mL solution once daily. Control animals received a vehicle, whereastreated animals were given 3a or RRD-238a at the indicated doses. Thetumor volumes were determined by measuring the length (l) and the width(w) and calculating the volume (V=lw²/2) as described previously (Sun99). Statistical significance between control and treated animals wereevaluated using Student's t-test.

Immunohistochemistry staining. Upon termination of xenograft anti-tumorexperiments, tumors were removed and fixed in 10% neutral-bufferedformalin before processing into paraffin blocks. Tissue sections (5micrometers (μm) thick) were cut from the blocks and stained with Ki-67,CD31, TUNEL, and phospho-Rb antibodies. Paraffin sections wererehydrated to PBS and processed using the following protocols. Sectionswere rinsed in dH₂O, and then subjected to microwave ‘antigen retrieval’for 20 minutes on 70% power, with a 1 minute cooling period after every5 minutes, in 0.01 M sodium citrate, pH 6.0 (Janssen P J, Brinkmann A O,Boersma W J, Van der Kwast T H. J Histochem Cytochem 1994;42(8):1169-75; Shi S R, Key M E, Kalra K L. J Histochem Cytochem 1991;39(6):741-748). Sections were cooled for 20 minutes, rinsed 3 times indH₂O, twice in PBS and incubated in 5% normal goat serum for 30 minutes.Sections were incubated in primary antibody for 1 hour in 5% normal goatserum, rinsed 3 times in PBS. For color development the slides weretreated with ABC kit (Vector Labs, Burlingame, Calif.) rinsed in dH₂O,and developed using DAB as chromogen. After a final rinse in dH₂O,sections were lightly counterstained in hematoxylin, dehydrated, clearedand coverslipped. Tissue sections were stained with hematoxylin andeosin (H&E) using standard histological techniques. Tissue sections werealso subjected to immunostaining for CD31 (BD Biosciences, San Diego,Calif., USA) using the avidin-biotin peroxidase complex technique. Mousemonoclonal antibody was used at 1:50 dilution following microwaveantigen retrieval (four cycles of 5 min each on high in 0.1 M citratebuffer). Apoptotic cells were detected using DeadEnd Colorimetric TUNELsystem (Promega, Madison, Wis.).

EXAMPLE 1 Identification, Design, and Synthesis of Disclosed Compounds

A screen was developed to identify small molecule inhibitors of theRb:Raf-1 interaction. A diversity set, comprising 1981 compounds(National Cancer Institute, Bethesda, Md.) was examined using aglutathione S-transferase-retinoblastoma/glutathione S-transferase-Raf-1kinase Enzyme-Linked ImmunoSorbent Assay screen (GST-Rb/GST-Raf-1ELISA). Two structurally related compounds (1) and (2) were discoveredthat strongly inhibited the Rb:Raf-1 interaction at a concentration of20 μM (100% for 1 and 95% for 2):

A library of small molecule inhibitors of Rb:Raf-1 binding based on thehits 1 and 2 were designed, synthesized, and assessed for biologicalactivity.

To determine the effects of various phenyl substituents on the abilityto inhibit Rb:Raf-1 binding, several benzylisothiourea derivatives 3,lacking substitution at the α benzylic position, were prepared in goodyields by reaction of thiourea with the appropriate benzyl halide(Scheme 1, Table 2). (Yong 1997) This method allowed us to rapidlygenerate a small library of benzylisothioureas, since a number ofsubstituted benzyl halides are commercially available. When notcommercially available the desired benzyl halides were obtained from thecorresponding benzyl alcohols prepared when necessary by NaBH₄ reductionof the corresponding aldehyde) followed by reaction with thionylchloride to generate the corresponding benzyl chloride. Thecorresponding benzylisothiourea derivatives 3 were usually obtained ingood to quantitative yields.

To evaluate the importance of the benzylic position to activity, thebenzylisothiouronium derivatives 4 bearing an alkyl group at thebenzylic position were prepared by the reaction of thiourea with theappropriate α-substituted benzyl halides (Scheme 1, Table 1). (Yong1997) The α-substituted benzyl halides were prepared by addition of analkylmagnesium bromide to the appropriate benzaldehyde, followed bytreatment of the intermediate alcohol with thionyl chloride.

The aryl methyl-, and hetero aryl methyl-thioureas 5 were obtained inmoderate to good yields from thiourea and the appropriate benzyl halide(Scheme 1, Table 3) in a similar fashion to the benzylisothioureaderivatives 3.

To determine the importance of the isothiourea sulfur atom, thebenzylguanidinium salts 6 (Scheme 1, Table 4) were obtained via thereaction between di-tert-butoxycarbonyl thiourea and the appropriatebenzylamine, (Yong 1997) followed by deprotection of the correspondingdi-tert-butoxycarbonyl guanidine product with tin(IV) chloride (Miel1997) or trifluoroacetic acid, (Guisado 2002) in moderate to goodyields. The condensation of α-halo ketones and thiourea, ormethylthiourea, was carried out efficiently under microwave irradiationconditions to afford good to quantitative yields of aminothiazoliumsalts of type 7 (Scheme 2, Table 5). Similarly, the condensation ofα-halo ketones and dimethylthiourea under microwave conditions affordedgood yields of the aminothiazolium quaternary salts of type 8 (Scheme 2,Table 6). A further set of analogues of the isothioureas was obtained byreaction of various thio-functionalized heterocycles(3-mercaptotriazole, 5-amino-3-mercaptotriazole,2-mercaptobenzoimidazole, 2-mercaptoimidazole, and4,5-dihydro-2-mercaptoimidazole) with 4-chlorobenzyl chloride or2,4-dichlorobenzyl chloride. The corresponding products 9a-d wereobtained in good to quantitative yield (Scheme 3, Table 7).

EXAMPLE 2

General Procedure for the Synthesis of Compounds 3a-t: A 10 milliliter(mL) microwave reaction tube was charged with the benzyl halide (1.0millimole, mmol) and thiourea (76 mg, 1.0 mmol) in ethanol (1.5 mL). Thetube was capped and irradiated in the microwave reactor (single-mode CEMDiscover™ system, CEM, Matthews, N.C.) at 100° C. for 15 minutes. Thesolid was filtered and solid washed with cold ethanol. The solid productwas dried under high vacuum to give the product.

EXAMPLE 3

General Procedure for the Synthesis of Compounds 7a-j and 8a-e: A 10 mLmicrowave reaction tube was charged with a mixture of the appropriateα-haloacetophenone (1.0 mmol) and thiourea (76 mg, 1.0 mmol) in ethanol(2.0 mL). The tube was capped and irradiated in the microwavesynthesizer actor (single-mode CEM Discover™ system) at 120° C. for 10minutes. The solid formed was filtered on a sintered funnel, washed withcold ethyl acetate and finally dried in vacuo.Compound Analytical Data

3a—White solid, mp 222-223° C.; ¹H NMR (400 MHz, d₆-DMSO) δ 4.58 (s,2H), 7.47 (dd, J=8.0 and 2.0 Hz, 1H), 7.63 (d, J=8.0 Hz, 1H), 7.70 (d,J=2.0 Hz, 1H), 9.31 (br s, 2H), 9.39 (br s, 2H); ¹³C NMR (100 MHz,d₆-DMSO) δ 32.6, 128.5, 130.0, 132.5, 133.3, 134.5, 135.1, 169.4; MS(ESI) m/z 235.0 (100%, [M+H]⁺); HRMS calcd for C₈H₉Cl₂N₂S: 234.9858;observed: 234.9854; HPLC analysis (Alltech C18): 90% methanol, 10%acetonitrile, flow rate 0.5 mL/min: t_(R) 3.26 min. 90% acetonitrile,10% water, flow rate 0.75 mL/min: t_(R) 2.05 min. 100% methanol, flowrate 0.5 mL/min: t_(R) 3.05 min.

3b—White solid, mp 199-200° C. ¹H NMR (400 MHz, d₆-DMSO) δ δ 4.61 (s,2H), 7.34-7.39 (m, 2H), 7.49-7.51 (m, 1H), 7.60-7.63 (m, 1H), 9.38 (brs, 2H), 9.46 (br s, 2H); MS (ESI) m/z 201.0 (100%, [M+H]⁺); HRMS calcdfor C₈H₁₀ClN₂S: 201.0247; observed: 201.0250; HPLC analysis (AlltechC18): 90% methanol, 10% acetonitrile, flow rate 0.5 mL/min: t_(R) 2.85min; 90% acetonitrile, 10% water, flow rate 0.75 mL/min: t_(R) 1.86 min.

3c—Light yellow solid, mp 203-204° C. ¹H NMR (400 MHz, d₆-DMSO) δ 4.42(s, 2H), 7.23 (d, J=8.0 Hz, 1H), 7.33 (d, J=8.0 Hz, 1H), 7.47 (t, J=8.0Hz, 1H); MS (ESI) m/z 219.0 (100%, [M+H]⁺); HRMS calcd for C₈H₉ClFN₂S:219.0153; observed: 219.0157; HPLC analysis (Alltech C18): 90% methanol,10% acetonitrile, flow rate 0.5 mL/min: t_(R) 2.90 min; 90%acetonitrile, 10% water, flow rate 0.75 mL/min: t_(R) 2.08 min.

3d—White solid, mp 253-254° C. ¹H NMR (400 MHz, d₆-DMSO) δ 4.64 (s, 2H),7.41 (t, J=8.0 Hz, 1H), 7.58 (dd, J=8.0 and 1.6 Hz, 1H), 7.67 (dd, J=8.0and 1.6 Hz, 1H), 9.31 (br s, 4H); MS (ESI) m/z 234.9 (100%, [M+H]⁺);HRMS calcd for C₈H₉Cl₂N₂S: 234.9858; observed: 234.9859; HPLC analysis(Alltech C18): 90% methanol, 10% acetonitrile, flow rate 0.5 mL/min:t_(R) 3.08 min; 90% acetonitrile, 10% water, flow rate 0.75 mL/min:t_(R) 2.05 min.

3e—White solid, mp 191-192° C. ¹H NMR (400 MHz, d₄-MeOH) δ 4.42 (s, 2H),7.35-7.37 (m, 3H), 7.46 (s, 1H); MS (ESI) m/z 201.0 (100%, [M+H]⁺); HRMScalcd for C₈H₁₀ClN₂S: 201.0247; observed: 201.0248; HPLC analysis(Alltech C18): 90% methanol, 10% acetonitrile, flow rate 0.5 mL/min:t_(R) 2.85 min; 90% acetonitrile, 10% water, flow rate 0.75 mL/min:t_(R) 2.01 min.

3f—Pale yellow solid, mp 163-164° C. ¹H NMR (250 MHz, d₄-MeOH) δ 4.49(s, 2H), 7.64 (d, J=8.0 Hz, 1H), 7.67 (dd, J=8.0 and 2.0 Hz, 1H), 7.86(d, J=2.0 Hz, 1H); MS (ESI) m/z 269.0 (100%, [M+H]⁺); HRMS calcd forC₉H₉ClF₃N₂S: 269.0121; observed: 269.0123; HPLC analysis (Alltech C18):90% methanol, 10% acetonitrile, flow rate 0.5 mL/min: t_(R) 3.18 min;90% acetonitrile, 10% water, flow rate 0.75 mL/min: t_(R) 2.11 min.

3g—White plates, mp 171-172° C. ¹H NMR (400 MHz, d₆-DMSO) δ 4.57 (s,2H), 7.40 (dd, J=6.8 and 1.6 Hz, 2H), 7.46 (dd, J=6.8 and 1.6 Hz, 2H),9.33 (br s, 2H), 9.46 (br s, 2H); ¹³C NMR (100 MHz, d₆-DMSO) 39.7, 129.3(2C), 131.5 (2C), 133.3, 135.3, 169.6; MS (ESI) m/z 201.0 (100%,[(M+H)]⁺); HRMS calcd for C₈H₁₀N₂SCl: 201.0247; observed: 201.0247; HPLCanalysis (Alltech C18): 90% methanol, 10% acetonitrile, flow rate 0.5mL/min: t_(R) 3.03 min; 90% acetonitrile, 10% water, flow rate 0.75mL/min: t_(R) 1.95 min.

3h—White solid, mp 240-241° C. ¹H NMR (400 MHz, d₆-DMSO) δ 4.39 (s, 2H),7.34 (dd, J=8.0 and 2.0 Hz, 1H), 7.53 (d, J=8.0 Hz, 1H), 7.60 (d, J=2.0Hz, 1H); MS (ESI) m/z 234.9 (100%, [M+H]⁺); HRMS calcd for C₈H₉Cl₂N₂S:234.9858; observed: 234.9860; HPLC analysis (Alltech C18): 90% methanol,10% acetonitrile, flow rate 0.5 mL/min: t_(R) 2.83 min; 90%acetonitrile, 10% water, flow rate 0.75 mL/min: t_(R) 1.88 min.

3i—White solid, mp 218-219° C. ¹H NMR (400 MHz, d₆-DMSO) δ 4.49 (s, 2H),7.37 (dd, J=8.4 and 2.0 Hz, 1H), 7.46 (d, J=8.4 Hz, 1H), 7.55 (d, J=2.0Hz, 1H); MS (ESI) m/z 234.9 (100%, [M+H]⁺); HRMS calcd for C₈H₉Cl₂N₂S:234.9858; observed: 234.9859; HPLC analysis (Alltech C18): 90% methanol,10% acetonitrile, flow rate 0.5 mL/min: t_(R) 2.80 min; 90%acetonitrile, 10% water, flow rate 0.75 mL/min: t_(R) 1.98 min.

3j—White solid, mp 233-234° C. ¹H NMR (400 MHz, d₄-MeOH) δ 4.67 (s, 2H),7.91 (d, J=8.0 Hz, 1H), 8.01 (d, J=8.0 Hz, 1H), 8.03 (s, 1H); MS (ESI)m/z 303.0 (100%, [M+H]⁺); HRMS calcd for C₁₀H₉F₆N₂S: 303.0391; observed:303.0391; HPLC analysis (Alltech C18): 90% methanol, 10% acetonitrile,flow rate 0.5 mL/min: t_(R) 2.66 min; 90% acetonitrile, 10% water, flowrate 0.75 mL/min: t_(R) 2.83 min.

3k—White solid, mp 234-235° C. ¹H NMR (400 MHz, d₄-MeOH) δ 1.33 (s, 9H),4.43 (s, 2H), 7.39 (d, J=11.0 Hz, 2H), 7.43 (d, J=11.0 Hz, 2H); ¹³C NMR(100 MHz, d₆-DMSO) 31.7 (3C), 34.4, 34.9, 126.2 (2C), 129.4 (2C), 132.7,151.0, 170.0; MS (ESI) m/z 241.2 (20%, [(M+NH₄)]⁺), 102.1 (100%). HRMScalcd for C₁₂H₁₉N₂S: 223.1263; observed: 223.1259; HPLC analysis(Alltech C18): 90% methanol, 10% acetonitrile, flow rate 0.5 mL/min:t_(R) 3.11 min; 90% acetonitrile, 10% water, flow rate 0.75 mL/min:t_(R) 2.08 min.

3l—Brown solid, mp 158-159° C. ¹H NMR (400 MHz, d₆-DMSO) δ 4.35 (s, 2H),5.96 (s, 2H), 6.80 (d, J=8.0 Hz, 1H), 6.88 (dd, J=8.0 and 1.6 Hz, 1H),6.90 (d, J=2.0 Hz, 1H); MS (ESI) m/z 211.0 (100%, [M+H]⁺), 135.0 (14%,[M−CH₃N₂S]⁺); HRMS calcd for C₉H₁₁N₂O₂S: 211.0535; observed: 211.0539;HPLC analysis (Alltech C18): 90% methanol, 10% acetonitrile, flow rate0.5 mL/min: t_(R) 2.93 min; 90% acetonitrile, 10% water, flow rate 0.75mL/min: t_(R) 1.95 min.

3m—White solid, mp 227-228° C. ¹H NMR (400 MHz, d₆-DMSO) δ 4.72 (s, 2H),7.71 (d, J=9.2 Hz, 2H), 8.19 (d, J=9.2 Hz, 2H), 9.32 (br s, 2H), 9.52(br s, 2H); ¹³C NMR (100 MHz, d₆-DMSO) δ 33.7, 124.4 (2C), 130.9 (2C),144.4, 147.6, 169.3; MS (ESI) m/z 212.0 (100%, [M+H]⁺); HRMS calcd forC₈H₁₀O₂N₃S: 212.0488; observed: 212.0489; HPLC analysis (Alltech C18):90% methanol, 10% acetonitrile, flow rate 0.5 mL/min: t_(R) 3.00 min;90% acetonitrile, 10% water, flow rate 0.75 mL/min: t_(R) 1.93 min.

3n—Colorless solid, mp 206-207° C. ¹H NMR (400 MHz, d₄-MeOH) δ 4.54 (s,2H), 7.39 (d, J=8.2 Hz, 2H), 7.43 (d, J=8.2 Hz, 2H); MS (ESI) m/z 235.0(100%, [M+H]⁺); HRMS calcd for C₉H₁₁F₃N₂S: 235.0511; observed: 235.0512;HPLC analysis (Alltech C18): 90% methanol, 10% acetonitrile, flow rate0.5 mL/min: t_(R) 2.80 min; 90% acetonitrile, 10% water, flow rate 0.75mL/min: t_(R) 1.85 min.

3o—White solid, mp 148-149° C. ¹H NMR (400 MHz, d₆-DMSO) δ 4.49 (s, 2H),6.89 (d, J=4.8 Hz, 2H), 7.34 (d, J=4.8 Hz, 2H), 9.35 (br s, 4H); ¹³C NMR(100 MHz, d₆-DMSO) δ 34.5, 55.8, 114.8 (2 C), 127.0, 131.0 (2 C), 159.5,170.0; MS (ESI) m/z 197.0 (18%, [M+H]⁺), 121.0 (100%, [M−CH₃N₂S]⁺); HRMScalcd for C₉H₁₃ON₂S: 197.0743; observed: 197.0733; HPLC analysis(Alltech C18): 90% methanol, 10% acetonitrile, flow rate 0.5 mL/min:t_(R) 3.06 min; 90% acetonitrile, 10% water, flow rate 0.75 mL/min:t_(R) 1.96 min.

3p—Colorless solid, mp 161-162° C. ¹H NMR (400 MHz, d₄-MeOH) δ 2.35 (s,3H), 4.41 (s, 2H), 7.21 (d, J=8.0 Hz, 2H), 7.34 (d, J=8.0 Hz, 2H); MS(ESI) m/z 181.0 (100%, [M+H]⁺); HRMS calcd for C₉H₁₃N₂S: 181.0794;observed: 181.0793; HPLC analysis (Alltech C18): 90% methanol, 10%acetonitrile, flow rate 0.5 mL/min: t_(R) 3.03 min; 90% acetonitrile,10% water, flow rate 0.75 mL/min: t_(R) 2.00 min.

3q—Light brown solid, mp 172-173° C. ¹H NMR (400 MHz, d₄-MeOH) δ 3.73(s, 3H), 3.82 (s, 6H), 4.37 (s, 2H), 6.71 (s, 2H); MS (ESI) m/z 181.1(100%, [M−CH₃N₂S]⁺); HRMS calcd for C₁₁H₁₇N₂O₃S: 257.0954; observed:257.0958; HPLC analysis (Alltech C18): 90% methanol, 10% acetonitrile,flow rate 0.5 mL/min: t_(R) 2.66 min; 90% acetonitrile, 10% water, flowrate 0.75 mL/min: t_(R) 4.21 min.

3r—White solid, mp 185-186° C. ¹H NMR (400 MHz, d₆-DMSO) δ 4.49 (s, 2H),7.30-7.42 (m, 5H), 9.22 (br s, 4H); MS (ESI) m/z 167.0 (100%, [M+H]⁺);HRMS calcd for C₈H₁₁N₂S: 167.0637; observed: 167.0643; HPLC analysis(Alltech C18): 90% methanol, 10% acetonitrile, flow rate 0.5 mL/min:t_(R) 2.98 min; 90% acetonitrile, 10% water, flow rate 0.75 mL/min:t_(R) 1.80 min.

3s—White solid, mp 231-232° C. ¹H NMR (400 MHz, d₆-DMSO) δ 4.67 (s, 2H),7.42 (t, J=8.0 Hz, 1H), 7.54 (d, J=8.0 Hz, 2H), 9.51 (br s, 4H); ¹³C NMR(100 MHz, d₆-DMSO) 32.2, 129.7, 130.3, 132.0, 135.8, 169.9; MS (ESI) m/z235.0 (100%, [M+H]⁺); HRMS calcd for C₈H₉Cl₂N₂S: 234.9858; observed:234.9855; HPLC analysis (Alltech C18): 90% methanol, 10% acetonitrile,flow rate 0.5 mL/min: t_(R) 3.08 min. 90% acetonitrile, 10% water, flowrate 0.75 mL/min: t_(R) 2.08 min.

3t—White solid, mp 265-266° C. ¹H NMR (400 MHz, d₆-DMSO) δ 4.64 (s, 2H),7.79 (s, 2H), 9.45 (br s, 4H); MS (ESI) m/z 270.9 (100%, [M+H]⁺); HRMScalcd for C₈H₈Cl₃N₂S: 268.9468; observed: 268.9469; HPLC analysis(Alltech C18): 90% methanol, 10% acetonitrile, flow rate 0.5 mL/min:t_(R) 2.23 min; 90% acetonitrile, 10% water, flow rate 0.75 mL/min:t_(R) 1.15 min.

4a—Off-white solid, mp 153-154° C. ¹H NMR (400 MHz, d₆-DMSO) δ 1.71 (d,J=6.8 Hz, 3H), 5.37 (q, J=6.8 Hz, 1H), 7.53 (dd, J=8.8 and 2.4 Hz, 1H),7.69 (d, J=8.8 Hz, 1H), 7.72 (d, J=2.4 Hz, 1H), 9.18 (br s, 2H), 9.35(br s, 2H); MS (ESI) m/z 248.9 (100%, [M+H]⁺); HRMS calcd forC₉H₁₁Cl₂N₂S: 249.0014; observed: 249.0017; HPLC analysis (Alltech C18):90% methanol, 10% acetonitrile, flow rate 0.5 mL/min: t_(R) 3.26 min;90% acetonitrile, 10% water, flow rate 0.75 mL/min: t_(R) 2.18 min.

4b—Beige solid, mp 108-109° C. ¹H NMR (400 MHz, d₄-MeOH) δ 1.70 (d,J=7.2 Hz, 3H), 4.94 (q, J=7.2 Hz, 1H), 7.40 (dd, J=8.0 and 1.8 Hz, 1H),7.56 (d, J=8.0 Hz, 1H), 7.64 (d, J=1.8 Hz, 1H); MS (ESI) m/z 249.0(100%, [M+H]⁺); HRMS calcd for C₉H₁₁Cl₂N₂S: 249.0014; observed:249.0017; HPLC analysis (Alltech C18): 90% methanol, 10% acetonitrile,flow rate 0.5 mL/min: t_(R) 2.68 min; 90% acetonitrile, 10% water, flowrate 0.75 mL/min: t_(R) 1.83 min.

4c—Yellow semisolid. ¹H NMR (400 MHz, d₆-DMSO) δ 0.85 (t, J=7.2 Hz, 3H),2.08 (sept, J=7.2 Hz, 2H), 5.24 (t, J=7.2 Hz, 1H), 6.85-7.25 (br s, 4H),7.53 (dd, J=8.4 and 2.0 Hz, 1H), 7.64 (d, J=8.4 Hz, 1H), 7.72 (d, J=2.0Hz, 1H); MS (ESI) m/z 263.0 (100%, [M+H]⁺); HRMS calcd for C₁₀H₁₃Cl₂N₂S:263.0176; observed: 263.0179; HPLC analysis (Alltech C18): 90% methanol,10% acetonitrile, flow rate 0.5 mL/min: t_(R) 3.15 min; 90%acetonitrile, 10% water, flow rate 0.75 mL/min: t_(R) 2.11 min.

4d—Off-white solid, mp 184-185° C. ¹H NMR (400 MHz, d₄-MeOH) δ 6.25 (s,1H), 7.31-7.45 (m, 6H), 7.46-7.53 (m, 4H); MS (ESI) m/z 243.1 (100%,[M+H]⁺); HRMS calcd for C₁₄H₁₅N₂S: 243.0950; observed: 243.0953; HPLCanalysis (Alltech C18): 90% methanol, 10% acetonitrile, flow rate 0.5mL/min: t_(R) 2.88 min; 90% acetonitrile, 10% water, flow rate 0.75mL/min: t_(R) 1.94 min.

4e—White solid, mp>300° C. (dec.). ¹H NMR (400 MHz, d₄-MeOH) δ 3.32 (d,J=7.6 Hz, 2H), 5.59 (t, J=7.6 Hz, 1H), 7.13-7.26 (m, 5H), 7.38 (dd,J=8.4 and 2.4 Hz, 1H), 7.43 (d, J=2.4 Hz, 1H), 7.67 (d, J=8.4 Hz, 1H);MS (ESI) m/z 325.0 (100%, [M+H]⁺); HRMS calcd for C₁₅H₁₅Cl₂N₂S:325.0327; observed: 325.0328; HPLC analysis (Alltech C18): 90% methanol,10% acetonitrile, flow rate 0.5 mL/min: t_(R) 3.23 min; 90%acetonitrile, 10% water, flow rate 0.75 mL/min: t_(R) 2.18 min.

5a—Off-white solid, mp 232-233° C. ¹H NMR (250 MHz, d₄-MeOH) δ 4.85 (s,2H), 7.60-7.73 (m, 3H), 7.95 (d, J=8.2 Hz, 2H), 8.33 (d, J=8.2 Hz, 2H);MS (ESI) m/z 295.0 (100%, [M+H]⁺); HRMS calcd for C₁₂H₁₂BrN₂S: 294.9899;observed: 294.9896; HPLC analysis (Alltech C18): 90% methanol, 10%acetonitrile, flow rate 0.5 mL/min: t_(R) 3.21 min; 90% acetonitrile,10% water, flow rate 0.75 mL/min: t_(R) 2.36 min.

5b—Yellow solid, mp 210-211° C. ¹H NMR (400 MHz, d₄-MeOH) δ 4.84 (s,2H), 8.15 (d, J=6.4 Hz, 2H), 8.87 (d, J=6.4 Hz, 2H); MS (ESI) m/z 168.0(100%, [M+H]⁺); HRMS calcd for C₇H₁₀N₃S: 168.0589; observed: 168.0592;HPLC analysis (Alltech C18): 90% methanol, 10% acetonitrile, flow rate0.5 mL/min: t_(R) 2.93 min; 90% acetonitrile, 10% water, flow rate 0.75mL/min: t_(R) 1.96 min.

5c—Off-white solid, mp 237-238° C. ¹H NMR (400 MHz, d₄-MeOH) δ 4.93 (s,2H), 7.57 (d, J=8.0 Hz, 1H), 7.61 (d, J=8.0 Hz, 1H), 7.70-7.73 (m, 2H),8.19-8.21 (m, 1H), 8.33-8.36 (m, 1H); MS (ESI) m/z 251.0 (100%, [M+H]⁺);HRMS calcd for C₁₂H₁₂ClN₂S: 251.0404; observed: 251.0405; HPLC analysis(Alltech C18): 90% methanol, 10% acetonitrile, flow rate 0.5 mL/min:t_(R) 1.71 min; 90% acetonitrile, 10% water, flow rate 0.75 mL/min:t_(R) 1.78 min.

5d—Brown solid, mp 204-205° C. ¹H NMR (400 MHz, d₄-MeOH) □4.83 (s, 2H),7.79-7.85 (m, 1H), 7.88-7.92 (m, 1H), 7.99-8.04 (m, 1H), 8.14-8.18 (m,2H), 7.37 (dd, J=8.0 and 5.6 Hz, 1H) □MS (ESI) m/z 218.0 (100%, [M+H]⁺);HRMS calcd for C₁₁H₁₂N₃S: 218.0746; observed: 218.0751; HPLC analysis(Alltech C18): 90% methanol, 10% acetonitrile, flow rate 0.5 mL/min:t_(R) 2.98 min; 90% acetonitrile, 10% water, flow rate 0.75 mL/min:t_(R) 2.00 min.

5e—Off-white solid, mp 222-223° C. ¹H NMR (400 MHz, d₆-DMSO) δ 4.68 (s,2H), 7.90-7.94 (m, 3H), 8.18-8.21 (m, 3H), 8.26 (d, J=1.2 Hz, 1H), 9.01(br s, 2H), 9.25 (br s, 2H); MS (ESI) m/z 297.0 (100%, [M+H]⁺); HRMScalcd for C₁₆H₁₃O₂N₂S: 297.0692; observed: 297.0693; HPLC analysis(Alltech C18): 90% methanol, 10% acetonitrile, flow rate 0.5 mL/min:t_(R) 4.83 min; 90% acetonitrile, 10% water, flow rate 0.75 mL/min:t_(R) 3.00 min.

5f—Yellow solid, mp 217-218° C. ¹H NMR (400 MHz, d₆-DMSO) δ 5.61 (s,2H), 7.54 (t, J=7.2 Hz, 2H), 7.65 (td, J=7.2 and 1.2 Hz, 2H), 8.11 (d,J=8.8 Hz, 2H), 8.44 (d, J=8.8 Hz, 2H), 8.66 (s, 1H), 9.52 (br s, 2H);¹³C NMR (100 MHz, d₆-DMSO) δ 29.1, 124.1, 124.5 (2C), 126.2 (2C), 127.8(2C), 129.5, 129.9 (2C), 130.6 (2C), 131.6 (2C), 170.5; MS (ESI) m/z191.1 (100%, [M−CH₃N₂S]⁺); HRMS calcd for C₁₆H₁₅N₂S: 267.0950; observed:267.0951.

6a—White solid, mp 220-221° C.; ¹H NMR (250 MHz, d₄-MeOH) δ 4.39 (s,2H), 7.37 (s, 4H); MS (ESI) m/z 184.0 (100%, [M+H]⁺); HRMS calcd forC₈H₁₁ClN₃: 184.0636; observed: 184.0638; HPLC analysis (Alltech C18):90% methanol, 10% acetonitrile, flow rate 0.5 mL/min: t_(R) 3.85 min.90% acetonitrile, 10% water, flow rate 0.75 mL/min: t_(R) 2.03 min.

6b—Off-white solid, mp 170-171° C.; ¹H NMR (400 MHz, d₆-DMSO) δ 4.42 (d,J=10.0 Hz, 2H), 7.19-7.85 (br s, 2H), 7.35 (s, 4H), 7.47 (d, J=6.0 Hz,1H), 7.93 (t, J=10.0 Hz, 1H); MS (ESI) m/z 184.0 (100%, [M+H]⁺); HRMScalcd for C₈H₁₁ClN₃: 184.0636; observed: 184.0638; HPLC analysis(Alltech C18): 90% methanol, 10% acetonitrile, flow rate 0.5 mL/min:t_(R) 3.13 min. 90% acetonitrile, 10% water, flow rate 0.75 mL/min:t_(R) 2.06 min.

6c—Pale yellow solid, mp 202-203° C.; ¹H NMR (400 MHz, d₆-DMSO) δ 4.43(d, J=10.0 Hz, 2H), 7.34 (d, J=8.4 Hz, 1H), 7.47 (dd, J=8.4 and 2.0 Hz,1H), 7.64 (d, J=2.0 Hz, 1H), 7.91 (t, J=6.0 Hz, 1H); MS (ESI) m/z 218.0(100%, [M+H]⁺); HRMS calcd for C₈H₁₀Cl₂N₃: 218.0246; observed: 218.0248;HPLC analysis (Alltech C18): 90% methanol, 10% acetonitrile, flow rate0.5 mL/min: t_(R) 3.01 min. 90% acetonitrile, 10% water, flow rate 0.75mL/min: t_(R) 2.03 min.

6d—Off-white solid, mp 188-189° C.; ¹H NMR (400 MHz, CDCl₃) δ 4.35 (s,2H), 7.13-7.35 (m, 4H), 7.40 (s, 1H), 7.61 (br s, 2H), 9.08 (br s, 2H);MS (ESI) m/z 184.0 (100%, [M+H]⁺), 126.0 (70%, [M−CH₃N₃]⁺); HRMS calcdfor C₈H₁₁ClN₃: 184.0636; observed: 184.0634.

6e—Colorless solid, mp 124-125° C.; ¹H NMR (400 MHz, d₄-MeOH) δ 4.49 (s,2H), 7.34-7.41 (m, 3H), 7.45-7.48 (m, 1H); MS (ESI) m/z 184.0 (100%,[M+H]⁺); HRMS calcd for C₈H₁₁ClN₃: 184.0636; observed: 184.0640; HPLCanalysis (Alltech C18): 90% methanol, 10% acetonitrile, flow rate 0.5mL/min: t_(R) 3.10 min. 90% acetonitrile, 10% water, flow rate 0.75mL/min: t_(R) 1.88 min.

6f—Pale yellow solid, mp 76-77° C.; ¹H NMR (400 MHz, d₄-MeOH) δ 4.38 (s,2H), 7.31 (d, J=8.4 Hz, 2H), 7.39 (d, J=8.4 Hz, 2H); MS (ESI) m/z 184.0(100%, [M+H]⁺); HRMS calcd for C₈H₁₁ClN₃: 184.0636; observed: 184.0634;HPLC analysis (Alltech C18): 90% methanol, 10% acetonitrile, flow rate0.5 mL/min: t_(R) 3.11 min. 90% acetonitrile, 10% water, flow rate 0.75mL/min: t_(R) 2.06 min.

7a—Off-white solid, mp 75-76° C.; ¹H NMR (400 MHz, d₆-DMSO) δ 2.92 (s,3H), 7.02 (s, 1H), 7.17 (t, J=9.2 Hz, 1H), 7.35 (t, J=9.8 Hz, 1H),7.82-7.86 (m, 1H); MS (ESI) m/z 227.0 (100%, [M+H]⁺); HRMS calcd forC₁₀H₉F₂N₂S: 227.0449; observed: 227.0448; HPLC analysis (Alltech C18):90% methanol, 10% acetonitrile, flow rate 0.5 mL/min: t_(R) 4.23 min.90% acetonitrile, 10% water, flow rate 0.75 mL/min: t_(R) 2.90 min.

7b—White solid, mp 185-186° C.; ¹H NMR (400 MHz, d₄-MeOH) δ 7.03 (s,1H), 7.50 (dd, J=8.2 and 2.0 Hz, 1H), 7.57 (d, J=8.2 Hz, 1H), 7.68 (d,J=2.0 Hz, 1H); MS (ESI) m/z 244.9 (100%, [M+H]⁺); HRMS calcd forC₉H₇Cl₂N₂S: 244.9701; observed: 244.9701; HPLC analysis (Alltech C18):90% methanol, 10% acetonitrile, flow rate 0.5 mL/min: t_(R) 4.65 min.90% acetonitrile, 10% water, flow rate 0.75 mL/min: t_(R) 3.05 min.

7c—Off-white solid, mp 112-113° C.; ¹H NMR (400 MHz, d₆-DMSO) δ 2.94 (s,3H), 7.10 (s, 1H), 7.25 (t, J=8.8 Hz, 2H), 7.82 (dd, J=8.8 and 5.6 Hz,2H); MS (ESI) m/z 209.0 (100%, [M+H]⁺); HRMS calcd for C₁₀H₁₀FN₂S:209.0543; observed: 209.0572; HPLC analysis (Alltech C18): 90% methanol,10% acetonitrile, flow rate 0.5 mL/min: t_(R) 4.48 min. 90%acetonitrile, 10% water, flow rate 0.75 mL/min: t_(R) 4.11 min.

7d—Yellow plates, mp 100-101° C.; ¹H NMR (400 MHz, d₆-DMSO) δ 2.97 (s,3H), 7.16 (s, 1H), 7.36 (t, J=7.2 Hz, 1H), 7.43 (t, J=7.2 Hz, 2H), 7.74(d, J=7.2 Hz, 2H); MS (ESI) m/z 191.0 (100%, [M+H]⁺); HRMS calcd forC₁₀H₁₁N₂S: 191.0637; observed: 191.0640; HPLC analysis (Alltech C18):90% methanol, 10% acetonitrile, flow rate 0.5 mL/min: t_(R) 4.58 min.90% acetonitrile, 10% water, flow rate 0.75 mL/min: t_(R) 3.00 min.

7e—White solid, mp 110-111° C.; ¹H NMR (400 MHz, d₆-DMSO) δ 7.08 (d,J=2.0 Hz, 1H), 7.23 (td, J=9.2 and 2.4 Hz, 1H), 7.40-7.46 (m, 1H),7.84-7.90 (m, 1H); MS (ESI) m/z 213.0 (100%, [M+H]⁺); HRMS calcd forC₉H₇F₂N₂S: 213.0293; observed: 213.0299; HPLC analysis (Alltech C18):90% methanol, 10% acetonitrile, flow rate 0.5 mL/min: t_(R) 3.93 min.90% acetonitrile, 10% water, flow rate 0.75 mL/min: t_(R) 2.68 min.

7f—White needles, mp 209-210° C.; ¹H NMR (400 MHz, d₆-DMSO) δ 2.97 (s,3H), 7.21 (s, 1H), 7.48 (dt, J=8.4 and 2.0 Hz, 2H), 7.82 (dt, J=8.4 and2.0 Hz, 2H); MS (ESI) m/z 225.0 (100%, [M+H]⁺); HRMS calcd forC₁₀H₁₀ClN₂S: 225.0247; observed: 225.0248; HPLC analysis (Alltech C18):90% methanol, 10% acetonitrile, flow rate 0.5 mL/min: t_(R) 5.15 min.90% acetonitrile, 10% water, flow rate 0.75 mL/min: t_(R) 3.31 min.

7g—Pale yellow solid, mp 134-135° C.; ¹H NMR (400 MHz, d₄-MeOH) δ 3.12(s, 3H), 7.04 (s, 1H), 7.50 (dd, J=8.8 and 2.0 Hz, 1H), 7.58 (d, J=8.8Hz, 1H), 7.69 (d, J=2.0 Hz, 1H); MS (ESI) m/z 258.9 (100%, [M+H]⁺); HRMScalcd for C₁₀H₉Cl₂N₂S: 258.9858; observed: 258.9858; HPLC analysis(Alltech C18): 90% methanol, 10% acetonitrile, flow rate 0.5 mL/min:t_(R) 4.90 min. 90% acetonitrile, 10% water, flow rate 0.75 mL/min:t_(R) 3.26 min.

7h—White solid, mp 219-220° C.; ¹H NMR (400 MHz, d₆-DMSO) δ 7.15 (s,1H), 7.29 (t, J=8.8 Hz, 2H), 7.40-7.46 (dd, J=8.4 and 5.2 Hz, 2H); MS(ESI) m/z 195.0 (100%, [M+H]⁺); HRMS calcd for C₉H₈FN₂S: 195.0386;observed: 195.0390; HPLC analysis (Alltech C18): 90% methanol, 10%acetonitrile, flow rate 0.5 mL/min: t_(R) 3.45 min. 90% acetonitrile,10% water, flow rate 0.75 mL/min: t_(R) 2.36 min.

7i—White solid, mp 211-212° C.; ¹H NMR (400 MHz, d₆-DMSO) δ 7.25 (s,1H), 7.52 (d, J=8.4 Hz, 2H), 7.78 (d, J=8.4, 2H); MS (ESI) m/z 211.0(100%, [M+H]⁺); HRMS calcd for C₉H₈ClN₂S: 211.0091; observed: 211.0092;HPLC analysis (Alltech C18): 90% methanol, 10% acetonitrile, flow rate0.5 mL/min: t_(R) 4.23 min. 90% acetonitrile, 10% water, flow rate 0.75mL/min: t_(R) 2.78 min.

7j—White solid, mp 200-201° C.; ¹H NMR (400 MHz, d₆-DMSO) δ 3.52 (br s,2H), 7.15 (s, 1H), 7.35-7.39 (m, 1H), 7.40-7.46 (m, 2H), 7.74 (dd, J=6.8Hz and 1.2 Hz, 1H); MS (ESI) m/z 177.0 (100%, [M+H]); HRMS calcd forC₉H₉N₂S: 177.0481; observed: 177.0479; HPLC analysis (Alltech C18): 90%methanol, 10% acetonitrile, flow rate 0.5 mL/min: t_(R) 3.90 min. 90%acetonitrile, 10% water, flow rate 0.75 mL/min: t_(R) 2.56 min.

8a—White solid, mp 212-213° C.; ¹H NMR (400 MHz, d₆-DMSO) δ 3.02 (s,3H), 3.44 (s, 3H), 7.12 (s, 1H), 7.54 (d, J=8.8 Hz, 2H), 7.61 (J=8.8 Hz,2H), 10.40 (s, 1H); MS (ESI) m/z 239.0 (100%, [M]⁺); HRMS calcd forC₁₁H₁₂ClN₂S: 239.0404; observed: 239.0404; HPLC analysis (Alltech C18):90% methanol, 10% acetonitrile, flow rate 0.5 mL/min: t_(R) 3.01 min.90% acetonitrile, 10% water, flow rate 0.75 mL/min: t_(R) 2.01 min.

8b—Off-white solid, mp 233-234° C.; ¹H NMR (400 MHz, d₆-DMSO) δ 3.02 (s,3H), 3.44 (s, 3H), 7.08 (s, 1H), 7.49-7.56 (m, 5H), 10.40 (s, 1H); MS(ESI) m/z 205.0 (100%, [M]⁺); HRMS calcd for C₁₁H₁₃N₂S: 205.0794;observed: 205.0797; HPLC analysis (Alltech C18): 90% methanol, 10%acetonitrile, flow rate 0.5 mL/min: t_(R) 3.01 min. 90% acetonitrile,10% water, flow rate 0.75 mL/min: t_(R) 1.96 min.

8c—White solid, mp 203-204° C.; ¹H NMR (400 MHz, d₆-DMSO) δ 3.02 (s,3H), 3.43 (s, 3H), 7.09 (s, 1H), 7.38 (t, J=8.8 Hz, 2H), 7.57 (m, 2H),10.42 (s, 1H); MS (ESI) m/z 223.0 (100%, [M]⁺); HRMS calcd forC₁₁H₁₂FN₂S: 223.0700; observed: 223.0707; HPLC analysis (Alltech C18):90% methanol, 10% acetonitrile, flow rate 0.5 mL/min: t_(R) 3.00 min.90% acetonitrile, 10% water, flow rate 0.75 mL/min: t_(R) 2.01 min.

8d—White solid, mp 253-254° C.; ¹H NMR (400 MHz, d₆-DMSO) δ 3.02 (s,3H), 3.36 (s, 3H), 7.22 (s, 1H), 7.29 (td, J=8.4 and 2.0 Hz, 1H), 7.52(td, J=10.0 and 2.0 Hz, 1H), 7.29 (qd, J=6.8 and 2.0 Hz, 1H), 10.40 (s,1H); MS (ESI) m/z 241.0 (100%, [M]⁺); HRMS calcd for C₁₁H₁₁ClF₂N₂S:241.0605; observed: 241.0618; HPLC analysis (Alltech C18): 90% methanol,10% acetonitrile, flow rate 0.5 mL/min: t_(R) 3.00 min. 90%acetonitrile, 10% water, flow rate 0.75 mL/min: t_(R) 1.98 min.

8e—Off-white solid, mp 271-272° C.; ¹H NMR (400 MHz, CDCl₃) δ 3.19 (s,3H), 3.70 (s, 3H), 6.56 (s, 1H), 7.30 (d, J=8.0 Hz, 2H), 7.43 (dd, J=8.0and 2.0 Hz, 2H), 7.59 (d, J=2.0 Hz, 2H); MS (ESI) m/z 273.0 (100%,[M]⁺); HRMS calcd for C₁₁H₁₀Cl₂N₂S: 273.0014; observed: 273.0016; HPLCanalysis (Alltech C18): 90% methanol, 10% acetonitrile, flow rate 0.5mL/min: t_(R) 3.01 min. 90% acetonitrile, 10% water, flow rate 0.75mL/min: t_(R) 2.03 min.

9a—White solid, mp 169-170° C. ¹H NMR (400 MHz, d₆-DMSO) δ 4.39 (s, 2H),7.34 (d, J=8.0 Hz, 1H), 7.49 (d, J=8.0 Hz, 2H), 7.61 (s, 1H), 8.45 (s,1H); ¹³C NMR (100 MHz, d₆-DMSO) 33.6, 128.0, 129.6, 132.9, 133.5, 134.8,135.2, 146.8, 156.7; MS (ESI) m/z 259.9 (100%, [M+H]⁺); HRMS calcd forC₉H₈Cl₂N₃S: 259.9810; observed: 259.9812; HPLC analysis (Alltech C18):90% methanol, 10% acetonitrile, flow rate 0.5 mL/min: t_(R) 4.56 min.90% acetonitrile, 10% water, flow rate 0.75 mL/min: t_(R) 2.81 min.

9b—White solid, mp 110-111° C.; ¹H NMR (400 MHz, d₆-DMSO) δ 4.31 (s,2H), 7.37 (d, J=8.0 Hz, 1H), 7.48 (d, J=8.0 Hz, 1H), 7.63 (s, 1H); MS(ESI) m/z 274.9 (100%, [M+H]⁺); HRMS calcd for C₉H₉Cl₂N₄S: 274.9919;observed: 274.9922; HPLC analysis (Alltech C18): 90% methanol, 10%acetonitrile, flow rate 0.5 mL/min: t_(R) 3.78 min. 90% acetonitrile,10% water, flow rate 0.75 mL/min: t_(R) 2.41 min.

9c—White solid, mp 161-162° C.; ¹H NMR (400 MHz, d₆-DMSO) δ 4.31 (s,2H), 3.32 (d, J=8.8 Hz, 2H), 3.36 (d, J=8.8 Hz, 2H), 8.44 (s, 1H); MS(ESI) m/z 226.0 (100%, [M+H]⁺); HRMS calcd for C₉H₉ClN₃S: 226.0200;observed: 226.0201; HPLC analysis (Alltech C18): 90% methanol, 10%acetonitrile, flow rate 0.5 mL/min: t_(R) 3.96 min. 90% acetonitrile,10% water, flow rate 0.75 mL/min: t_(R) 3.43 min.

9d—Pale yellow solid, mp 122-123° C.; ¹H NMR (400 MHz, d₆-DMSO) δ 4.32(s, 2H), 7.36 (s, 4H); MS (ESI) m/z 241.0 (100%, [M+H]⁺); HRMS calcd forC₉H₁₀ClN₄S: 241.0309; observed: 241.0313; HPLC analysis (Alltech C18):90% methanol, 10% acetonitrile, flow rate 0.5 mL/min: t_(R) 3.56 min.90% acetonitrile, 10% water, flow rate 0.75 mL/min: t_(R) 2.35 min.

9e—White solid, mp 183-184° C.; ¹H NMR (250 MHz, CDCl₃) δ 4.35 (s, 2H),7.09-7.11 (m, 2H), 7.13 (s, 2H), 7.38 (m, 1H); MS (ESI) m/z 258.9 (100%,[M+H]); HRMS calcd for C₁₀H₉Cl₂N₂S: 258.9858; observed: 258.9854; HPLCanalysis (Alltech C18): 90% methanol, 10% acetonitrile, flow rate 0.5mL/min: t_(R) 4.00 min. 90% acetonitrile, 10% water, flow rate 0.75mL/min: t_(R) 2.66 min.

9f—White solid, mp 137-138° C.; ¹H NMR (250 MHz, CDCl₃) δ 4.17 (s, 2H),7.09 (s, 2H), 7.11 (d, J=8.5 Hz, 2H), 7.24 (d, J=8.5 Hz, 2H); MS (ESI)m/z 225.0 (100%, [M+H]⁺); HRMS calcd for C₁₀H₁₀ClN₂S: 225.0247;observed: 225.0251; HPLC analysis (Alltech C18): 90% methanol, 10%acetonitrile, flow rate 0.5 mL/min: t_(R) 3.81 min. 90% acetonitrile,10% water, flow rate 0.75 mL/min: t_(R) 2.53 min.

9g—White solid, mp 167-168° C.; ¹H NMR (250 MHz, d₆-DMSO) δ 3.84 (s,4H), 4.66 (s, 2H), 7.45 (d, J=8.6 Hz, 2H), 7.54 (d, J=8.6 Hz, 2H), 10.68(s, 2H); MS (ESI) m/z 227.0 (100%, [M+H]⁺); HRMS calcd for C₁₀H₁₂ClN₂S:227.0404; observed: 227.0410; HPLC analysis (Alltech C18): 90% methanol,10% acetonitrile, flow rate 0.5 mL/min: t_(R) 2.78 min. 90%acetonitrile, 10% water, flow rate 0.75 mL/min: t_(R) 1.70 min.

9h—Pale pink solid, mp 121-122° C.; ¹H NMR (250 MHz, CDCl₃) δ 4.84 (s,1H), 7.34 (dt, J=8.5 and 2.0 Hz, 2H), 7.48 (dt, J=8.5 and 2.0 Hz, 2H),7.49 (td, J=7.5 and 1.2 Hz, 1H), 7.60 (td, J=7.5 and 1.2 Hz, 1H), 7.82(dd, J=7.5 and 1.2 Hz, 1H), 8.27 (dd, J=7.5 and 1.2 Hz, 1H); MS (ESI)m/z 292.0 (100%, [M+NH₄]⁺); HPLC analysis (Alltech C18): 90% methanol,10% acetonitrile, flow rate 0.5 mL/min: t_(R) 7.35 min. 90%acetonitrile, 10% water, flow rate 0.75 mL/min: t_(R) 6.70 min.

9i—White solid, mp 223-224° C.; ¹H NMR (250 MHz, d₆-DMSO) δ 3.87 (s,4H), 4.76 (s, 2H), 7.49 (dd, J=8.3 and 2.1 Hz, 1H), 7.73 (d, J=2.1 Hz,1H), 7.80 (d, J=8.3 Hz, 1H), 10.83 (s, 2H); MS (ESI) m/z 262.0 (100%,[M+H]⁺); HRMS calcd for C₁₀H₁₁Cl₂N₂S: 261.0014; observed: 261.0014; HPLCanalysis (Alltech C18): 90% methanol, 10% acetonitrile, flow rate 0.5mL/min: t_(R) 3.01 min. 90% acetonitrile, 10% water, flow rate 0.75mL/min: t_(R) 1.98 min.

9j—Off-white solid, mp 124-125° C.; ¹H NMR (400 MHz, CDCl₃) δ 4.91 (s,2H), 7.22 (dd, J=8.2 and 2.1 Hz, 1H), 7.44 (t, J=8.0 Hz, 2H), 7.56 (t,J=8.3 Hz, 1H), 7.68 (d, J=8.3 Hz, 1H), 7.80 (d, J=8.0 Hz, 1H), 8.19 (d,J=8.1 Hz, 1H); MS (ESI) m/z 325.9 (100%, [M+NH₄]⁺); HPLC analysis(Alltech C18): 90% methanol, 10% acetonitrile, flow rate 0.5 mL/min:t_(R) 8.26 min. 90% acetonitrile, 10% water, flow rate 0.75 mL/min:t_(R) 7.86 min.

The following additional compounds were made by the preceding methods:

EXAMPLE 4 The Disclosed Compounds Have Rb:Raf-1 Binding InhibitionActivity

Compounds that can directly inhibit GST Rb binding to GST-Raf-1 wereidentified from the NCI diversity library of 1981 compounds as describedunder Methods, resulting in two compounds, (1) and (2), which inhibitedRb:Raf-1 binding 100% and 95% respectively. Both were benzyl-isothioureaderivatives of similar structure (FIG. 1 a). Next, the library ofcompounds, comprising the benzylisothiouronium derivatives 3a-t, 4a-eand 5a-f, of the benzylguanidinium derivatives 6a-f, of theaminothiazolium derivatives 7a-j, and 8a-e; and of the mercaptoanalogues 9a-j was screened for Rb:Raf-1 binding inhibitory propertiesusing the GST-Rb/GST-Raf-1 ELISA assay. The results are reported as %inhibition of Rb:Raf-1 binding at a concentration of 20 micromolar (μM,Tables 1-7). A dose response was performed for compounds able to inhibitthe interaction by 80% or greater at 20 μM. The results, shown in Table8, are reported as the concentration required to disrupt the interactionby 50% relative to an untreated control (IC₅₀ value).

Generally, the activities related to the aromatic substitution pattern,where the halogenated derivatives exhibited highest potencies. For thebenzylisothiouronium derivatives 3a-t, active compounds tended topossess a monosustituted or disubstituted benzene ring, bearing at leastone halide in either one of the positions ortho, meta, or para to thecarbon bound to the isothiouronium group. The sensitivity to halidesubstitution is shown by comparison of 3b (100% inhibition), 3g (88%inhibition) and 3r (inactive). The presence of either 2- or 4-chlorosubstituent strongly affects the activity. The activity of 3a suggestedthat the activity of 1 derives from the presence of thebenzylisothiouronium ion and not the nitrophenolate ion.

Most of the chlorine-containing derivatives display IC₅₀ values in thesubmicromolar range (3b 0.575, 3c 0.081, 3e 0.230 and 3f 0.312 μM). The2,4-dichloro aromatic substitution pattern, which is common to 3a and tothe hit (1), particularly tended to enhance the inhibitory activity(IC₅₀ values: 3a, 77 nM; 1, 100 nM). By contrast, derivative 3r, having2 hydrogens in place of the 2 chlorines of 3a or 1 tended to beinactive. Thus, the chlorines tend to increase the compound's ability todisrupt the binding of Rb to Raf-1 (Tables 1 and 8). Two chlorines onthe phenyl ring tended to be better one as 3a was 3 to 6-fold morepotent than 3b (2-chlorobenzylisothiourea, IC₅₀=575 nM), 3e(3-chlorobenzylisothiourea, IC₅₀−230 nM) and 3g(4-chlorobenzylisothiourea, IC₅₀=274 nM). Placement of the chlorinestends to affect activity strongly because the 2,3-dichloro derivative 3dwas more than 2-fold less active compared to 3a (IC₅₀=164 nM),3,4-dichloro derivative 3h was 50 times less active compared 3a(IC₅₀=3900 nM) and the 2,6-dichloro derivative 3s and 2,4,6-trichloroderivative 3t were inactive compared to 3a. Furthermore, replacing the 2chloro groups in 3a by 2 trifluoromethyl groups as in the2,4-trifluoromethyl derivative 3j tended to reduce the activity (Table 1and 8). A decrease in potency was observed when the compounds weresubstituted in the alpha position with alkyl groups (Table 2). Forexample the addition of the methyl or ethyl group to 3a results in afour and seven fold weaker inhibition respectively (IC₅₀ 3a 77 nM; 4aIC₅₀ 322 nM; IC₅₀ 4c 567 nM).

The highest inhibitory activity among the arymethylisothiouroniumderivatives 5a-f was displayed by the 1-bromonaphthyl derivative 5a (80nM). Substituting a bromo by a chloro and linking to the isothiourea atthe 4 position as in 3c tended to reduce activity by 24-fold (Table 3and 8) as shown by the analogue 5c (IC₅₀=1900 nM).

The isothiourea group tended to increase activity as replacing theisothiourea in 3a by guanidinium as in 6c tended to dramatically reducethe activity (Table 4 and 8). The activities of the benzylguanidiniumderivatives 6a-f, were in some cases dependant on the counterion.Compound 6a, a benzylguanidinium hydrosulfate, tended to display thehighest potency (IC₅₀ 539 nM; 100% inhibition at 20 μM), whereas thebenzylguanidinium hydrochlorides 6b-d were less active (59-61%inhibition at 20 μM), and the benzylguanidinium trifluoroacetates 6e and6f tended to be the least active of the series (respectively, 25 and 16%inhibition at 20 μM).

The aminothiazolium derivatives 7a-j generally displayed modestactivity, as did most of the analogues 8a-e. The most active compoundswas the difluoro derivative 7a, which inhibited the Rb:Raf-1 binding 53%at 20 μM. Finally, amongst the thioheterocyclic analogues 9a-j thehighest potencies were displayed by the triazoles 9a (97 nM) and 9b (131nM), which both have the 2,4-dichloro aromatic substitution pattern.These derivatives differ by one amino group, which tended to have lesseffect on activity. TABLE 1 Structures, yields of benzylisothiouroniumsalts 3a-t, and inhibition of Rb:Raf-1 binding.

Com- % Inhibition pound R² R³ R⁴ R⁵ R⁶ X Yield (%) at 20 μM 3a Cl H Cl HH Cl 98 100 3b Cl H H H H Cl 76 100 3c H F Cl H H Cl 96 100 3d Cl Cl H HH Cl 99 100 3e H Cl H H H Cl 90 100 3f H CF₃ Cl H H Cl 81 94 3g H H Cl HH Cl 87 88 3h H Cl Cl H H Cl 83 81 3i Cl H H Cl H Cl 93 79 3j CF₃ H CF₃H H Br 86 53 3k H H tBu H H Cl 81 52 3l H O—CH₂—O H H Cl 95 49 3m H HNO₂ H H Cl 75 46 3n H H CF₃ H H Br 77 46 3o H H OMe H H Cl 98 45 3p H HMe H H Cl 65 44 3q H OMe OMe OMe H Cl 52 20 3r H H H H H Cl 78 0 3s Cl HH H Cl Cl 81 0 3t Cl H Cl H Cl Cl 75 0

TABLE 2 Structures, yields of substituted benzylisothiouronium salts4a-e, and inhibition of Rb:Raf-1

Compounds R R² R³ R⁴ Yield (%) % Inhibition at 20 μM 4a Me Cl H Cl 91100 4b Me H Cl Cl 89 99 4c Et Cl H Cl 77 100 4d Ph H H H 51 21 4e CH₂PhCl H Cl 18 32

TABLE 3 Structures, yields of aryl- and heteroaryl-methylisothiouroniumsalts 5a-f, and inhibition of Rb:Raf-1 binding. Compounds Yield (%) %Inhibition at 20 μM 5a

82 100 5b

78 94 5c

10 93 5d

98 76 5e

86 56 5f

97 22

TABLE 4 Structures, yields of benzylguanidinium salts 6a-f, andinhibition of Rb:Raf-1 binding.

Compound R² R⁴ X Yield (%) % Inhibition at 20 μM 6a H Cl HSO₄ 32 100 6bH Cl Cl 45 61 6c Cl Cl Cl 87 61 6d Cl H Cl 95 59 6e Cl H CF₃CO₂ 58 25 6fH Cl CF₃CO₂ 61 16

TABLE 5 Structures, yields of aminothiazolium type salts 7a-j, andinhibition of Rb:Raf-1 binding.

Compound R R² R⁴ Yield (%) % Inhibition at 20 μM 7a Me F F 89 53 7b H ClCl 99 45 7c Me H F 99 43 7d Me H H 87 39 7e H F F 96 39 7f Me H Cl 99 347g Me Cl Cl 95 33 7h H H F 99 32 7i H H Cl 89 29 7j H H H 80 22

TABLE 6 Structures, yields of aminothiazolium type salts 8a-e, andinhibition of Rb:Raf-1 binding.

Compound R² R³ R⁴ Yield (%) % Inhibition at 20 μM 8a H H Cl 84 37 8b H HH 96 22 8c H H F 79 19 8d F H F 89 5 8e Cl H Cl 75 —

TABLE 7 Structures, yields of benzylthioimidazole type analogues 9a-j,and inhibition of Rb:Raf-1 binding.

% Inhibition Compound R R² Yield (%) at 20 μM 9a

Cl 99 92 9b

Cl 94 91 9c

H 90 81 9d

H 93 76 9e

Cl 21 68 9f

H 15 67 9g

H 84 47 9h

H 87 46 9i

Cl 91 45 9j

Cl 83 40

TABLE 8 Inhibition of the Rb:Raf-1 binding (IC₅₀) of the most activederivatives 3-9. Compound IC₅₀ (nM)^(a) 3a 77 ± 4 5a 80 ± 6 (1) 81 ± 43c  81 ± 10 9a 97 ± 4 9b 131 ± 22 3d 164 ± 9  3e 230 ± 25 3g 274 ± 24(2) 283 ± 46 3f 312 ± 53 4a 322 ± 87 4b  510 ± 116 6a 539 ± 13 4c 567 ±91 3b  575 ± 115 5c 1900 ± 40  3i 2110 ± 180 5b 2630 ± 330 3h  3900 ±2460^(a)Drug concentration that inhibits the Rb:Raf-1 binding by 50% . . . .Each drug concentration was tested in triplicate, and the standard errorof each value was less than 10%.

EXAMPLE 5 The Disclosed Compounds are Selective for Rb:Raf-1 OverRb-E2F1

The selectivity of (1) and (2) for inhibition of Rb:Raf-1 compared toRb-E2F1 was tested in a GST ELISA assay. Both hits were at least 200fold more selective for Rb:Raf-1 over Rb-E2F1 (FIG. 1 b).

EXAMPLE 6 The Disclosed Compounds Disrupt Rb:Raf-1 In Vitro

The ability of the small molecules to disrupt Rb:Raf-1 was confirmed byGST pull-down assays, as described in Methods. Asynchronous U937 lysateswere incubated with GST-Rb beads in the presence or absence of theselected compounds or an 8 amino acid Raf-1 peptide and the binding ofRaf-1 assessed by western blotting. It was found that presence of 20 μMof (1) (IC₅₀ of 81±4 nM), (2) (IC₅₀ of 283±46 nM) and the Raf-1 peptideinhibited the binding of Raf-1 to GST Rb beads (FIG. 1C).

EXAMPLE 7 The Disclosed Compounds Effectively Disrupt Rb:Raf-1 in IntactCells

U937 cells were serum starved for 48 hours and subsequently serumstimulated for 2 hours in the presence or absence of 20 μM of thecompounds. Both (1) and (2) inhibited the binding of Raf-1 to Rbsignificantly (IC₅₀ of 81±4 nM and 283±46 nM, respectively), as seen byimmunoprecipitation-Western blot analysis (FIG. 1 d); Raf-1 peptideconjugated to penetratin was used as a positive control. Thus it appearsthat these two compounds were capable of disrupting the Rb:Raf-1interaction in vitro and in cultured cells.

EXAMPLE 8 Disclosed Compound 3a Selectively Inhibits Rb:Raf-1 Binding

As shown in FIG. 1 a, (1) and (2) consist each of a benzyl-isothioureaderivative and a phenyl-based counter ion. To determine potency of thebenzyl-isothiourea group versus the phenyl counter ions, these compoundswere compared to 3a (RRD-251, FIG. 2 a), which had chloride as thecounter ion. (1) had an IC₅₀ of 81±4 nM and (2) had an IC₅₀ of 283±46nM, while 3a showed a value of 77±3.6 nM (FIG. 2 a). Next, theselectivity of the Rb:Raf-1 binding disruptors in vitro was determinedto be selective for Rb:Raf-1 over Rb/E2F1, Rb/HDAC1, Rb/prohibitin andRaf-1/Mek association (FIG. 2 b). FIG. 2 a shows that 3a is as effectiveas (1) at inhibiting Rb:Raf-1 binding in an ELISA assay. In addition,serum stimulation of binding of Rb to Raf-1 in A-549 cells was inhibitedby 3a and the Raf-1 peptide but not a scrambled peptide coupled topenetratin (FIG. 2 c, left panel). In contrast, the Rb binding to E2F1and serum-mediated binding of Raf-1 to Mek1/2 were not affected by 3afurther confirming the selectivity of this small molecule for Rb:Raf-1(FIG. 2 c left and middle panels). It was found that the level of cyclinD as well as its association with Rb was reduced by 3a (FIG. 2C, rightpanel). Examination of lysates from cells treated with 3a for 18 hoursshowed a marked reduction in Rb phosphorylation, as seen by westernblotting using an antibody to Rb or one that specifically recognizes Rbphosphorylated on serines 807 and 811; treatment of cells withBAY43-9006 (sorafenib) did not seem to have any effect on Rbphosphorylation (FIG. 2 d). Further, in vitro assays showed that 3a didnot affect the kinase activities associated with Raf-1, cyclin D orcyclin E (FIG. 2 e). These results suggest that the reduction in Rbphosphorylation in cells treated with 3a is due to a disruption in theassociation of Raf-1 with Rb and not due to an inhibition of the kinaseactivity of Raf-1.

Since serum is known to stimulate the binding of Raf-1 to Rb and leadsto the dissociation of the co-repressor Brg-1 from E2F-responsiveproliferative promoters like cdc6, cdc25 and TS promoters, it washypothesized that 3a may interfere with this process. Chromatinimmunoprecipitation assays demonstrated that Raf-1 binding to the abovepromoters upon serum-stimulation is disrupted by pre-treatment of cellswith 3a. Furthermore, the corresponding dissociation of the co-repressorBrg-1 from these promoters was also inhibited by 3a. This suggests that3a can modulate the transcriptional regulatory functions of Rb in thenucleus by modulating its phosphorylation status and affecting itsinteraction with chromatin remodeling proteins like Brg-1. Binding ofE2F1, HDAC1 and HP1 was not affected.

EXAMPLE 9 Compound 3a Inhibited Osteosarcoma Proliferation

Given the selectivity of 3a described in FIGS. 1 and 2 above fordisrupting the Rb:Raf-1 interaction, it was examined if 3a can inhibitthe proliferation of cells and whether such an inhibition required afunctional Rb gene. Two osteosarcoma cell lines, U2-OS (which has wildtype Rb) and Saos-2 (which is Rb null), were rendered quiescent by serumstarvation and subsequently stimulated with serum in the presence orabsence of 20 μM 3a for 18 hours and S-phase entry was assessed bymeasuring BrdU incorporation. 3a inhibited S-phase entry of U2-OS cells;but it had little effect on Saos-2 cells, suggesting that 3a inhibitscell proliferation in an Rb-dependent manner.

EXAMPLE 10 Compound 3a Inhibited Epithelial Lung Cancer CellProliferation

To further confirm that 3a requires a functional Rb to inhibit tumorcell proliferation, A549 cells (human epithelial lung carcinoma) werestably transfected with two different shRNA constructs (sh6 and sh8) toknock down Rb expression. As expected, A549 cells stably expressing theRb shRNAs had significantly less Rb protein compared to parental A549cells (FIG. 3 b). 3a was very effective at inhibiting S-phase entry inparental A549 cells but had no effect on cells stably expressing sh6 andsh8, which lacked Rb (FIG. 3 c). This result confirms that 3a arrestscell proliferation in a Rb dependent manner.

EXAMPLE 11 Compound 3a Inhibited Non-Small Cell Lung CarcinomaProliferation

Because many cancers contain more than one mutation in tumor suppressorgenes or oncogenes, we determined the ability of 3a to inhibitproliferation in cell lines containing alterations in key regulatorygenes. Compound 3a was able to inhibit 90% of S-phase entry in the H1650non-small cell lung cancer (NSCLC) cell line that carry mutations in thetyrosine kinase domain of EGFR (FIG. 3 d).

EXAMPLE 12 Compound 3a Inhibited Proliferation of 3 Pancreatic CancerCell Lines

Using the methods of the preceding examples, it was found that compound3a could inhibit S-phase entry by 50-65% in pancreatic cancer cells suchas Aspc1, PANC1, and CAPAN2 that harbor a non-functional p16INK4a gene(FIG. 3 d).

EXAMPLE 13 Compound 3a Inhibited Proliferation of Two Glioblastoma CellLines

Using the methods of the preceding examples, compound 3a also inhibitedS-phase entry of two glioblastoma cell lines U87MG and U251MG, both ofwhich are null for p16 and PTEN.

EXAMPLE 14 Compound 3a Inhibited Metastatic Breast Cancer CellProliferation

The metastatic human breast cancer cell line MDA-MB-231 harbors a K-Rasmutation and overexpresses EGFR. Using the methods of the precedingexamples, compound 3a inhibited MDA-MB-231 proliferation by 56% (FIG. 3d).

EXAMPLE 15 Compound 3a Inhibited Melanoma Cell Proliferation

A V600E mutation in the B-Raf oncogene is associated with 66% ofmelanomas and leads to an over-activation of phospho-Erk signaling. TheA375 melanoma cell line harbors the V600E B-Raf mutation. Using themethods of the preceding examples, 3a was inhibited 58% of S-phase entryin this cell line.

EXAMPLE 16 Compound 3a Inhibited Prostate Cancer Cell Proliferation

Prostate cell lines LNCaP and PC3 both contain mutations in K-Ras andPTEN genes. Using the methods of the preceding examples, compound 3ainhibited proliferation 86% and 35% respectively (FIG. 2 d). Theseresults indicate that disruption of Rb:Raf-1 interaction could inhibitthe proliferation of cell lines harboring a wide array of genemutations.

EXAMPLE 17 Anticancer Activity of Disclosed Compounds is Via Disruptionof Rb:Raf-1 Interaction

It was hypothesized that if compound 3a selectively targets the Rb:Raf-1interaction, the forced expression of a downstream target or Rb such asE2F1, but not of the upstream regulator Cyclin D, would rescue theanti-proliferative effects of compound 3a. To this end, A549 cells wereinfected with Ad-E2F1 or Ad-cyclin D, in the presence of 20 μM of 3a for36 h. Ad-GFP infected cells were used as a control. BrdU incorporationassays showed that ectopic expression of E2F1 efficiently overcame theanti-proliferative activity of 3a, whereas over-expression of cyclin Dhad only a partial effect (FIG. 3 e). Without wishing to be bound bytheory, these results provide support for the hypothesis that theassociation of Raf-1 with Rb may be needed for the complete inactivationof Rb by the kinases associated with cyclins D and E, and thus that theactivity of compound 3a in disrupting this association may lead to itsanticancer effects.

EXAMPLE 18 Disclosed Compounds Disrupt Angiogenesis

An experiment was performed to determine whether angiogenic tubuleformation could be inhibited by the disclosed compounds. Human aorticendothelial cells (HAECs) were grown in matrigel in the presence orabsence of 20 μM (1), (2), or 3a. It was found that while angiogenictubules formed in control wells, the disclosed disruptors of theRb:Raf-1 interaction significantly inhibited the angiogenic tubuleformation (FIG. 3 f).

EXAMPLE 19 Disclosed Compounds Inhibit Anchorage Independent TumorGrowth (Soft Agar)

Experiments were also carried out to examine the effect of 3a ininhibiting the adherence-independent growth of cancer cells in softagar. It was found that compound 3a significantly inhibited the growthof A549 (human epithelial lung carcinoma), H1650 (NSCLC), SK-MEL-5,SK-MEL-28 (melanoma), and PANC1 (pancreatic) cells in soft agar. Theability of 3a to inhibit cell proliferation, adherence-independentgrowth and angiogenesis demonstrates that it has properties desirable inanti-cancer drugs.

EXAMPLE 20 Compounds 3a & 9a Significantly Inhibited Human Tumor Line InVivo

Experiments were performed to assess whether compounds 3a and 9a couldinhibit human tumor growth in vivo using a nude mice xenograft model.Athymic nude mice were implanted with 1×10⁷ A549 cells bilaterally andthe tumors were allowed to reach 200 mm³ in size before treatment began.FIG. 4A shows that tumors from vehicle treated mice grew to an averagesize of 1039±128 mm³. In contrast, tumors treated with either 50milligrams per kilogram (mpk)/day (i.p.) or 150 mpk/day (oral) of 3a didnot grow (50 mpk: 144±20 mm³; 150 mpk 148±32 mm³). Similar results wereobserved with H1650 in that 3a (50 mpk/day) inhibited the growth ofthese tumors significantly (FIG. 4 b; 2185±326 mm³ in vehicle treatedanimals compared to 557±76 mm3 in 3a treated animals). Similarly,compound 9a (RRD-238a), an analogue of 3a, was equally effective ininhibiting the growth of A549 tumors in nude mice (FIG. 4 c).Furthermore, to examine whether 3a could affect the growth of tumorslacking Rb in vivo, sh6 and sh8 (shRNA for Rb) cell lines were implantedinto the flanks of nude mice. The mice were given 3a orally at 150 mpkand the control groups were administered the vehicle. Mice harboring SH6and SH8 tumors and treated with 3a did not respond, they continued togrow at the rate of the wild type A549 tumors (FIGS. 4 d, e).

At the end of the drug treatment, the A549 tumors were removed from themice and fixed in formalin or snap frozen in liquid nitrogen for furtherhistochemical analysis. The tumors were analyzed by immunohistochemistrystaining with hematoxylin and eosin (H&E), TUNEL, Ki-67, phospho-Rb(807,811), and CD-31. Histopathological analysis revealed a significantinhibition of proliferation as seen by a reduction in Ki-67 staining(FIG. 4 f). The tissues showed a reduction in phosphorylation of Rb asseen by staining with an antibody to phospho-Rb (FIG. 4 f). The tumorsalso showed a reduction in microvasculature, as seen by CD31 staining(FIG. 4 f). A dose dependent increase in apoptosis (TUNEL) was observedin tumors treated with 3a, probably as a result of inhibition ofangiogenesis (FIG. 4 f). Tumors were homogenized and lysates wereprepared to assess the inhibition of Rb:Raf-1 interaction in vivo. 3awas found to inhibit Rb:Raf-1 but not Rb/E2F1 interaction in the tumorxenografts (FIG. 4 g). To examine whether the sh6 and sh8 tumorsmaintained downregulation of Rb, lysates made from the sh6 and sh8tumors at the end of the experiment and a western blot was done for Rb,It was found that these tumors lacked Rb, further confirming thatcompound 3a specifically targets the Rb:Raf-1 protein interaction toinhibit cell proliferation and tumor growth (FIG. 4 h).

Discussion

The Ras/Raf/Mek/MAPK cascade is a proliferative pathway induced by awide array of growth factors and is activated in many human tumors. Ithas been shown that signaling pathways through the MAP kinase cascade donot proceed in a linear fashion, but rather that they have been found tohave substrates outside the cascade as well. Without wishing to be boundby theory, in this context, the Rb protein appears to be an importantcellular target of the Raf-1 kinase outside the MAP kinase cascade. Thebinding of Raf-1 to Rb was found to occur only in proliferating cellsand contributed to cell cycle progression. Further, it was found thatthe level of Rb:Raf-1 interaction was elevated in NSCLC tissue,suggesting that it may have contributed to the oncogenic process. Theseobservations support the hypothesis that targeting the Rb:Raf-1interaction with the disclosed compounds is a viable method to developanticancer drugs.

The cell-permeable, orally available, and target specific small moleculecompound 3a, can maintain the tumor suppressor functions of Rb. The invitro results indicate that compound 3a selectively inhibits theRb:Raf-1 interaction without targeting the binding partners of Rb andRaf-1, such as E2F1, prohibitin, HDAC1 and MEK1/2. Further, compound 3afunctions by inhibiting the interaction of Raf-1 and Rb withoutinhibiting Raf-1 kinase activity or the kinase activity associated withcyclins D or E. Also, compound 3a inhibited cell cycle and decreased thelevels of cyclin D while cdk activity was unaffected. Compound 3ademonstrated Rb dependence to inhibit cell cycle progression and tumorgrowth in cell lines. These results further confirm the specificity of3a for targeting Rb:Raf-1. Mice harboring A549 tumors responded totreatment with 3a administered by i.p. or oral gavage. Tumor tissuedisplayed a decrease in proliferation, Rb phosphorylation, andangiogenesis and an increase in apoptosis. Importantly, A-549 tumorswhere Rb was knockdown are resistant to 3a, further suggesting that 3ainhibits tumor growth by targeting the Rb:Raf-1 interaction.

These results show that the mechanism of 3a mediated growth arrest islikely by targeting the Rb:Raf-1 interaction. Aberrant signalingmechanisms surrounding the Ras/MAPK and Rb/E2F1 pathways are commonlypresent in cancers. The disclosed compounds, such as compound 3a, couldinhibit S-phase entry in potentially 35%-90% of all of the cell lines.Based on the substantial in vitro and in vivo results disclosed herein,it is believed that the disclosed compounds, in particular compound 3a,are excellent candidates for the treatment of cancer patients whosetumors harbor genetic aberrations that lead to inactivation of Rb byRaf-1.

REFERENCES

The entire teachings of each document cited herein, including thefollowing, are incorporated by reference.

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1. A compound represented by structural formula Ia:

and pharmaceutically acceptable salts and solvates thereof, wherein:each dashed line (- - -) represents a single bond, or one (- - -) is adouble bond; Ring A is optionally substituted, and zero, one, or two ofthe ring atoms in Ring A are N; Ring A is fused with zero, one, or twooptionally substituted 3-15 membered monocyclic or polycyclic ringsselected from the group consisting of aryl, heteroaryl, heterocyclyl,and cycloaliphatic; Y is an optionally substituted C₁₋₃ alkylene or C₁₋₃alkenylene; X¹ is independently —O—, —S—, or optionally substituted—CH₂—, —CH═, —NH—, or —N═, and X² and X³ are independently ═S, oroptionally substituted —NH₂, ═NH, or —SH, or an optionally substituted3-7 membered aryl, heteroaryl, heterocyclyl, or cycloaliphatic ring, or:X² and X³ are independently —S—, or optionally substituted —NH—, —N═, or═N—, and X² and X³ are linked α, β, or γ through an optionallysubstituted alkyl, alkenyl, heteroalkyl, heteroalkenyl, heteroatom,aryl, heteroaryl, heterocyclyl, or cycloaliphatic linking group, therebyforming an optionally substituted heteroaryl or heterocyclyl ring; or X²is independently —S— or optionally substituted —NH—, —N═, or ═N—, and X²is linked α, β, or γ to a carbon of Y through an optionally substitutedalkyl, alkenyl, heteroalkyl, heteroalkenyl, or heteroatom linking group,thereby forming an optionally substituted heteroaryl or heterocyclylring, and wherein X³ is optionally —H; each substitutable carbon isoptionally substituted with a carbon substituent independently selectedfrom the group consisting of —F, —Cl, —Br, —I, —CN, —NO₂, —R^(a),—OR^(a), —C(O)R^(a), —OC(O)R^(a), —C(O)OR^(a), —SR^(a), —C(S)R^(a),—OC(S)R^(a), —C(S)OR^(a), —C(O)SR^(a), —C(S)SR^(a), —S(O)R^(a),—SO₂R^(a), —SO₃R^(a), —OSO₂R^(a), —OSO_(a)R^(a), —PO₂R^(a)R^(b),—OPO₂R^(a)R^(b), —PO₃R^(a)R^(b), OPO₃R^(a)R^(b), —N(R^(a)R^(b)),—C(O)N(R^(a)R^(b)), —C(O)NR^(a)NR^(b)SO₂R^(c), —C(O)NR^(a)SO₂R^(c),—C(O)NR^(a)CN, —SO₂N(R^(a)R^(b)), —NR^(a)SO₂R^(b), —NR^(c)C(O)R^(a),—NR^(c)C(O)OR^(a), NR^(c)C(O)N(R^(a)R^(b)), —C(NR^(c))—N(R^(a)R^(b)),—NR^(d)—C(NR^(c))—N(R^(a)R^(b)), —NR^(a)N(R^(a)R^(b)),—CR^(c)═CR^(a)R^(b), —C≡CR^(a), ═O, ═S, ═CR^(a)R^(b), ═NR^(a), ═NOR^(a),and ═NNR^(a), or two substitutable carbons are linked with C₁₋₃alkylenedioxy; each substitutable nitrogen is: optionally substitutedwith a nitrogen substituent independently selected from the groupconsisting of —CN, —NO₂, —R^(a), —OR^(a), —C(O)R^(a), —C(O)R^(a)-aryl,—OC(O)R^(a), —C(O)OR^(a), —SR^(a), —S(O)R^(a), —SO₂R^(a), —SO₃R^(a),—N(R^(a)R^(b)), —C(O)N(R^(a)R^(b)), —C(O)NR^(a)NR^(b)SO₂R^(c),—C(O)NR^(a)SO₂R^(c), —C(O)NR^(a)CN, —SO₂N(R^(a)R^(b)), —NR^(a)SO₂R^(b),—NR^(c)C(O)R^(a), —NR^(c)C(O)OR^(a), —NR^(c)C(O)N(R^(a)R^(b)), andoxygen to form an N-oxide; and optionally protonated or quaternarysubstituted to carry a positive charge which is balanced by apharmaceutically acceptable counteriion; wherein each R^(a)-R^(d) isindependently —H, C₁₋₆ alkyl, C₁₋₆ alkoxy, C₁₋₆ haloalkyl, C₁₋₆haloalkoxy, C₁₋₆ aralkyl, aryl, heteroaryl, heterocyclyl, orcycloaliphatic, or, —N(R^(a)R^(b)), taken together, is an optionallysubstituted heterocyclic group; X², X³, each nitrogen substitutent, andeach carbon substituent is optionally protected with a protecting group;and provided the compound is not benzyl N,N′-bis(tert-butoxycarbonyloxy)carbamimidothioate or a salt represented by


2. The compound of claim 1, provided that the compound is not benzylcarbamimidothioate.
 3. The compound of claim 1, wherein one dashed linerepresents a double bond.
 4. The compound of claim 1, wherein the ringrepresented by Ring A is an optionally substituted phenyl, biphenyl,naphthyl, pyrenyl, anthracyl, 9,10-dihydroanthracyl, fluorenyl, pyridyl,pyrimidyl, pyrazinyl, triazinyl, quinolinyl, isoquinolinyl,quinazolinyl, napthyridyl, pyridopyrimidyl, benzothienyl,benzothiazolyl, benzoisothiazolyl, thienopyridyl, thiazolopyridyl,isothiazolopyridyl, benzofuranyl, benzooxazolyl, benzoisooxazolyl,furanopyridyl, oxazolopyridyl, isooxazolopyridyl, indolyl, isoindolyl,benzimidazolyl, benzopyrazolyl, pyrrolopyridyl, isopyrrolopyridyl,imidazopyridyl, or pyrazolopyridyl group.
 5. The compound of claim 4,wherein the ring represented by Ring A is an optionally substitutedphenyl, naphthyl, anthracyl, fluorenyl, 9,10-dihydroanthracyl, pyridyl,pyrimidyl, pyrazinyl, triazinyl, quinolinyl, isoquinolinyl,quinazolinyl, or napthyridyl group.
 6. The compound of claim 4, whereinthe ring represented by Ring A is an optionally substituted naphthyl,pyridyl, quinolinyl, or isoquinolinyl group, or a substituted phenylgroup.
 7. The compound of claim 6, wherein each substitutable carbon isoptionally substituted with —F, —Cl, —Br, —I, —CN, —NO₂, —R^(a),—OR^(a), —C(O)R^(a), —OC(O)R^(a), —C(O)OR^(a), —SR^(a), —SO₂R^(a),—SO₃R^(a), —OSO₂R^(a), —OSO₃R^(a), —N(R^(a)R^(b)), —C(O)N(R^(a)R^(b)),—C(O)NR^(a)NR^(b)SO₂R^(c), —C(O)NR^(a)SO₂R^(c), —SO₂N(R^(a)R^(b)),—NR^(a)SO₂R^(b), —NR^(c)C(O)R^(a), —NR^(c)C(O)OR^(a), ═S, or ═O, or twosubstitutable carbons are linked with C₁₋₃ alkylenedioxy.
 8. Thecompound of claim 7, wherein: one, two or three substitutable carbons inthe ring represented by Ring A are substituted with —F, —Cl, —Br, —I,—CN, —NO₂, C₁₋₆ alkyl, C₁₋₆ alkoxy, —CF₃, or C₁₋₆ haloalkoxy, or twosubstitutable carbons in the ring represented by Ring A are linked withC₁₋₂ alkylenedioxy, provided that at least one ring atom of Ring Aadjacent to the point of attachment of Ring A to the rest of thecompound is unsubstituted.
 9. The compound of claim 8, wherein: Ring Ais a six-membered ring that is monsubstituted at its 2, 3, or 4positions or disubstituted at its 2,3, 2,4, 2,5 or 3,4 positions withsubstituents independently selected from the group consisting of —F,—Cl, —Br, —NO₂, C₁₋₆ alkyl, —CF₃, and methylenedioxy; and the 1 positionis the point of attachment of Ring A to the rest of the compound. 10.The compound of claim 9, wherein Ring A is a phenyl independentlydisubstituted with —Br, —Cl, —F or —CF₃ at its 2,3, 2,4, or 2,5positions.
 11. The compound of claim 4, wherein Y is optionallysubstituted with —OH, C₁₋₆ alkyl, C₁₋₆ alkoxy, C₁₋₆ aralkyl, ═O, or ═S.12. The compound of claim 11, wherein Y is ethylene or methylene, and Yis unsubstituted or substituted with C₁₋₃ alkyl.
 13. The compound ofclaim 4, wherein the compound is represented by structural formula Ib:

wherein: R^(y) is —H, —OH, C₁₋₆ alkyl, C₁₋₆ alkoxy, C₁₋₆ haloalkyl, C₁₋₆haloalkoxy, or C₁₋₆ aralkyl, or —R^(y) is ═O or ═S; X¹ and X³ areindependently N or S; Z¹ is a heteroatom or a β- or γ-bonded alkyl,alkenyl, heteroalkyl, heteroalkenyl, aryl, heteroaryl, heterocyclyl, orcycloaliphatic linking group, whereby Ring B is an optionallysubstituted heterocyclyl or heteroaryl ring; and Ring B is optionallysubstituted at any substitutable ring atom with halogen, —CN, —NO₂,—R^(a), —OR^(a), —C(O)R^(a), —OC(O)R^(a), —C(O)OR^(a), —SR^(a),—SO₂R^(a), —SO₃R^(a), —OSO₂R^(a), —OSO₃R^(a), N(R^(a)R^(b)),—C(O)N(R^(a)R^(b)), —SO₂N(R^(a)R^(b)), —NR^(a)SO₂R^(b),—NR^(c)C(O)R^(a), —NR^(c)C(O)OR^(a), or ═O.
 14. The compound of claim13, wherein the compound is represented by structural formula Ic:


15. The compound of claim 14, wherein: Z¹ is 1,2-phenylene, —CH₂CH₂—,—CH═CH—, or —N═CH₂—; and Ring B is optionally substituted at anysubstitutable ring atom with halogen, —CN, —NO₂, —R^(a), —OR^(a),—N(R^(a)R^(b)), —C(O)N(R^(a)R^(b)), —NR^(c)C(O)R^(a), or ═O.
 16. Thecompound of claim 15, wherein Z¹ is —N═CH₂— optionally substituted withN(R^(a)R^(b)).
 17. The compound of claim 15, wherein the compound isselected from the group consisting of:


18. The compound of claim 4, wherein the compound is represented bystructural formula Id:

wherein R³, R³′, R^(y) and R² are independently —H, —OH, C₁₋₆ alkyl,C₁₋₆ alkoxy, C₁₋₆ haloalkyl, C₁₋₆ haloalkoxy, C₁₋₆ aralkyl, aryl,heteroaryl, heterocyclyl, or cycloaliphatic, or —R^(y) is ═O or ═S. 19.The compound of claim 18, wherein R^(y) is —H, C₁₋₆ alkyl, C₁₋₆ aralkyl,or ═O.
 20. The compound of claim 18, wherein the compound is selectedfrom the group consisting of:


21. The compound of claim 18, wherein the compound is selected from thegroup consisting of:


22. The compound of claim 18, wherein the compound is selected from thegroup consisting of:


23. The compound of claim 4, wherein the compound is represented bystructural formula Ie:

wherein R¹ is —H, —OH, C₁₋₆ alkyl, C₁₋₆ alkoxy, C₁₋₆ haloalkyl, C₁₋₆haloalkoxy, C₁₋₆ aralkyl, aryl, heteroaryl, heterocyclyl, orcycloaliphatic, or —R¹ represents a lone pair of the nitrogen to whichit is attached.
 24. The compound of claim 23, wherein the compound isrepresented by structural formula If:

wherein R³, R³′, R^(y) and R² are independently —H, —OH, C₁₋₆ alkyl,C₁₋₆ alkoxy, C₁₋₆ haloalkyl, C₁₋₆ haloalkoxy, C₁₋₆ aralkyl, aryl,heteroaryl, heterocyclyl, or cycloaliphatic, or —R^(y) is ═O or ═S. 25.The compound of claim 24, wherein R^(y) is —H, C₁₋₆ alkyl, or C₁₋₆aralkyl, or —R^(y) is ═O.
 26. The compound of claim 25, wherein thecompound is selected from the group consisting of:


27. The compound of claim 4, wherein the compound is represented bystructural formula Ig:

wherein: X³ is —NR³R³′ or optionally halogenated phenyl; R³, R³′, and R¹are independently —H, C₁₋₆ alkyl, C₁₋₆ alkanoyl or C₁₋₆ aralkanoyl, or—R¹ represents a lone pair of the nitrogen to which it is attached; Z isa β-bonded alkyl or alkenyl linking group, whereby Ring C is aheterocyclyl or heteroaryl ring; and Ring C is optionally substituted atany substitutable ring atom with halogen, —CN, —NO₂, —R^(a), —OR^(a),—N(R^(a)R^(b)), ═O, or phenyl.
 28. The compound of claim 27, wherein thecompound is selected from the group consisting of:


29. The compound of claim 27, wherein the compound is selected from thegroup consisting of:


30. The compound of claim 1, wherein the compound is represented bystructural formula Ih:

wherein R³, R³′, R¹ and R² are independently —H, —OH, C₁₋₆ alkyl, C₁₋₆alkoxy, or C₁₋₆ aralkyl, provided that at least two of R³, R³′, R^(y)and R² are —H; or R³ and R² together are —N═CH— or —CH═N—, optionallysubstituted with —N(R^(a)R^(b)) or —C(O)N(R^(a)R^(b)); the ringrepresented by Ring A is: an optionally substituted naphthyl, pyridyl,quinolinyl, or isoquinolinyl; or a phenyl that is monsubstituted at its2, 3, or 4 positions or disubstituted at its 2,3, 2,4, or 2,5 or 3,4positions, wherein the 1 position is the point of attachment of Ring Ato the rest of the compound; and substituents for Ring A areindependently —Br, —Cl, —F, —CN, —NO₂, C₁₋₆ alkoxy, methylenedioxy or—CF₃, provided that when Ring A is phenyl at least one substituent ofRing A is —F or —Cl.
 31. A method of synthesizing a compound representedby structural formula Ia:

comprising reacting a first reagent represented by structural formulaIa:

with a second reagent represented by the following structural formula:

thereby forming the compound, wherein: each dashed line (- - -)represents a single bond, or one (- - -) is a double bond; Ring A isoptionally substituted, and zero, one, or two of the ring atoms in RingA are N; Ring A is fused with zero, one, or two optionally substituted3-15 membered monocyclic or polycyclic rings selected from the groupconsisting of aryl, heteroaryl, heterocyclyl, and cycloaliphatic; Y andY′ are an optionally substituted C₁₋₃ alkylene or C₁₋₃ alkenylene; W isa leaving group or unsubstituted —NH₂; X¹′ is independently ═CH₂, —NH₂,═NH, —OH, —SH, ═O, or ═S, and X¹ is correspondingly —O—, —S—, oroptionally substituted —CH₂—, —CH═, —NH—, or —N═; wherein when W isunsubstituted —NH₂, X¹′ is and X¹ is optionally substituted —NH— or —N═;X²′ and X³′ are independently ═S or optionally substituted —NH₂, ═NH, or—SH, or an optionally substituted 3-7 membered aryl, heteroaryl,heterocyclyl, or cycloaliphatic ring, and X² and X³ correspond to X²′and X³′, respectively, or: X²′ and X³′ are independently —S— oroptionally substituted —NH—, —N═, or ═N—, and are optionally linked α,β, or γ through an optionally substituted alkyl, alkenyl, heteroalkyl,heteroalkenyl, heteroatom, aryl, heteroaryl, heterocyclyl, orcycloaliphatic linking group, thereby forming an optionally substitutedheteroaryl or heterocyclyl ring, wherein X² and X³ are likewised linkedto correspond to X²′ and X³′, respectively; or X²′ is independently ═Sor optionally substituted —NH₂, ═NH, or —SR^(a), X² is correspondingly—S— or optionally substituted —NH—, —N═, or ═N—, and X² is linked α, β,or γ to a carbon of Y through an optionally substituted alkyl, alkenyl,heteroalkyl, heteroalkenyl, or heteroatom linking group, thereby formingan optionally substituted heteroaryl or heterocyclyl ring, and whereinX³ is optionally —H; each substitutable carbon is optionally substitutedwith a carbon substituent independently selected from the groupconsisting of —F, —Cl, —Br, —I, —CN, —NO₂, —R^(a), —OR^(a), —C(O)R^(a),—OC(O)R^(a), —C(O)OR^(a), —SR^(a), —C(S)R^(a), —OC(S)R^(a), —C(S)OR^(a),—C(O)SR^(a), —C(S)SR^(a), —S(O)R^(a), —SO₂R^(a), —SO₃R^(a), —OSO₂R^(a),—OSO₃R^(a), —PO₂R^(a)R^(b), —OPO₂R^(a)R^(b), —PO₃R^(a)R^(b),—OPO₃R^(a)R^(b), —N(R^(a)R^(b)), —C(O)N(R^(a)R^(b)),—C(O)NR^(a)NR^(b)SO₂R^(c), —C(O)NR^(a)SO₂R^(c), —C(O)NR^(a)CN,—SO₂N(R^(a)R^(b)), —NR^(a)SO₂R^(b), —NR^(c)C(O)R^(a), —NR^(c)C(O)OR^(a),NR^(c)C(O)N(R^(a)R^(b)), —C(NR^(c))—N(R^(a)R^(b)),—NR^(d)—C(NR^(c))—N(R^(a)R^(b)), —NR^(a)N(R^(a)R^(b)),—CR^(c)═CR^(a)R^(b), —C≡CR^(a), ═O, ═S, ═CR^(a)R^(b), ═NR^(a), ═NOR^(a),and ═NNR^(a), or two substitutable carbons are linked with C₁₋₃alkylenedioxy; each substitutable nitrogen is: optionally substitutedwith a nitrogen substituent independently selected from the groupconsisting of —CN, —NO₂, —R^(a), —OR^(a), —C(O)R^(a), —C(O)R^(a)-aryl,—OC(O)R^(a), —C(O)OR^(a), —SR^(a), —S(O)R^(a), —SO₂R^(a), —SO₃R^(a),—N(R^(a)R^(b)), —C(O)N(R^(a)R^(b)), —C(O)NR^(a)NR^(b)SO₂R^(c),—C(O)NR^(a)SO₂R^(c), —C(O)NR^(a)CN, —SO₂N(R^(a)R^(b)), —NR^(a)SO₂R^(b),—NR^(c)C(O)R^(a), —NR^(c)C(O)OR^(a), —NR^(c)C(O)N(R^(a)R^(b)), andoxygen to form an N-oxide; and optionally protonated or quaternarysubstituted, carrying a positive charge which is balanced by apharmaceutically acceptable counteriion; wherein each R^(a)-R^(d) isindependently —H, C₁₋₆ alkyl, C₁₋₆ alkoxy, C₁₋₆ haloalkyl, C₁₋₆haloalkoxy, C₁₋₆ aralkyl, aryl, heteroaryl, heterocyclyl, orcycloaliphatic, or, —N(R^(a)R^(b)), taken together, is an optionallysubstituted heterocyclic group; X²′, X³′, X², X³, each nitrogensubstitutent, and each carbon substituent is optionally protected with aprotecting group; and provided the compound is not benzylN,N′-bis(tert-butoxycarbonyloxy) carbamimidothioate or a saltrepresented by


32. The method of claim 31, wherein W is halogen, optionally alkylatedhalonium ion, optionally alkylated oxonium ion, perchlorate,amonioalkanesulfonate, halosulfonate, haloalkyl sulfonate, alkylsulfonate, optionally substituted aryl sulfonate, or optionallysubstituted —N(arylsulfonate)₂.
 33. The method of claim 32, wherein W is—Cl, —Br, —I, —OSO₂F, —OSO₂CF₃, —OSO₂C₄F₉, —OSO₂CH₂CF₃, —OSO₂CH₃,tosylate, brosylate, nosylate, or —N(tosylate)₂.
 34. The method of claim32, wherein W is —Cl or —Br.
 35. The method of claim 32, wherein thefirst and second reagents are reacted together under microwaveirradiation.
 36. The method of claim 32, wherein Y and Y′ are optionallysubstituted with —OH, ═O, ═S, C₁₋₆ alkyl, C₁₋₆ alkoxy, C₁₋₆ haloalkyl,C₁₋₆ haloalkoxy, or C₁₋₆ aralkyl;
 37. The method of claim 32, wherein Yand Y′ is optionally substituted with —OH, ═O, ═S, C₁₋₆ alkyl, C₁₋₆alkoxy, or C₁₋₆ aralkyl;
 38. The method of claim 32, wherein Y and Y′ isethylene or methylene, optionally substituted with C₁₋₃ alkyl.
 39. Themethod of claim 32, wherein X¹′ is —OH, ═O, —SH, or ═S, and X¹ iscorrespondingly —O— or —S—.
 40. The method of claim 32, wherein X¹′ is—SH or ═S and X¹ is —S—.
 41. The method of claim 38, wherein the firstreagent is represented by structural formula IIb:

the second reagent is represented by structural formula IIIb:

and the compound is represented by structural formula Ib:

wherein: R^(y) and R^(y)′ are —H, —OH, C₁₋₆ alkyl, C₁₋₆ alkoxy, C₁₋₆haloalkyl, C₁₋₆ haloalkoxy, or C₁₋₆ aralkyl, or —R^(y) and —R^(y′) are═O or ═S; X³′ and X³ are N or S; X¹′ is —OH or —SH and X¹ iscorrespondingly —O— or —S—; Z¹ and Z¹′ are each a heteroatom or a β- orγ-bonded alkyl, alkenyl, heteroalkyl, heteroalkenyl, aryl, heteroaryl,heterocyclyl, or cycloaliphatic linking group, whereby Ring B and RingB′ are corresponding heterocyclyl or heteroaryl rings; and Ring B andRing B′ are optionally substituted at any substitutable ring atom withhalogen, —CN, —NO₂, —R^(a), —OR^(a), —C(O)R^(a), —OC(O)R^(a),—C(O)OR^(a), —SR^(a), —SO₂R^(a), —SO₃R^(a), —OSO₂R^(a), —OSO₃R^(a),—N(R^(a)R^(b)), —C(O)N(R^(a)R^(b)), —SO₂N(R^(a)R^(b)), —NR^(a)SO₂R^(b),—NR^(c)C(O)R^(a), —NR^(c)C(O)OR^(a), or ═O.
 42. The method of claim 40,wherein X¹′ is —SH and X¹ is correspondingly —S—.
 43. The method ofclaim 41, wherein: Z¹ and Z¹′ are each 1,2-phenylene, —CH₂CH₂—, —CH═CH—,or —N═CH₂—; and Ring B and Ring B′ are optionally substituted at anysubstitutable ring atom with halogen, —CN, —NO₂, —R^(a), —OR^(a),—N(R^(a)R^(b)), —C(O)N(R^(a)R^(b)), —NR^(c)C(O)R^(a), or ═O.
 44. Themethod of claim 42, wherein Z¹ and Z¹′ are —N═CH₂— optionallysubstituted with —N(R^(a)R^(b)).
 45. The method of claim 42, wherein thecompound is selected from the group consisting of:


46. The method of claim 38, wherein the second reagent is represented bystructural formula IIIc:

and the compound is represented by structural formula Id:

wherein R³, R³′, R^(y) and R² are independently —H, —OH, C₁₋₆ alkyl,C₁₋₆ alkoxy, C₁₋₆ haloalkyl, C₁₋₆ haloalkoxy, C₁₋₆ aralkyl, aryl,heteroaryl, heterocyclyl, or cycloaliphatic, or —R^(y) is ═O or ═S. 47.The method of claim 45, wherein R¹ is —H, C₁₋₆ alkyl, C₁₋₆ aralkyl, or═O.
 48. The method of claim 46, wherein the compound is selected fromthe group consisting of:


49. The method of claim 46, wherein the compound is selected from thegroup consisting of:


50. The method of claim 46, wherein the compound is selected from thegroup consisting of:


51. The method of claim 31, wherein: X¹′ is; none of X²′, X³′ X², or X³is unsubstituted —NH₂ or ═NH; and the first reagent is represented bystructural formula IIc:

whereby the compound is represented by structural formula Ie:

wherein: R¹ is —H, —OH, C₁₋₆ alkyl, C₁₋₆ alkoxy, C₁₋₆ haloalkyl, C₁₋₆haloalkoxy, C₁₋₆ aralkyl, aryl, heteroaryl, heterocyclyl, orcycloaliphatic, or —R¹ represents a lone pair of the nitrogen to whichit is attached; and one dashed line (- - -) represents a double bond andthe other dashed lines represent single bonds.
 52. The method of claim50, wherein: the second reagent is represented by structural formulaIIId:

wherein PG and PG′ are amine protecting groups thereby producing aprotected intermediate represented by structural formula:

wherein R¹ is —H, —OH, C₁₋₆ alkyl, C₁₋₆ alkoxy, C₁₋₆ haloalkyl, C₁₋₆haloalkoxy, C₁₋₆ aralkyl, aryl, heteroaryl, heterocyclyl, orcycloaliphatic, or —R¹ represents a lone pair of the nitrogen to whichit is attached.
 53. The method of claim 50, wherein the first reagentand the second reagent are reacted together in the presence of anoptionally substituted 1-alkyl-2-halopyridinium salt or an optionallysubstituted 1-aryl-2-halopyridinium salt.
 54. The method of claim 50,wherein the first reagent and the second reagent are reacted together inthe presence of 1-methyl-2-chloropyridinium iodide.
 55. The method ofclaim 51, further comprising removing protecting groups PG and PG′ fromthe protected intermediate, thereby producing the compound, representedby structural formula Ij:


56. The method of claim 52, wherein the protecting groups PG and PG′ areindependently t-butyloxycarbonyl or benzyloxycarbonyl.
 57. The method ofclaim 53, wherein the step of removing protecting groups PG and PG′ fromthe protected intermediate comprises contacting the protectedintermediate with trifluoracetic acid or SnCl₄.
 58. The method of claim52, wherein the compound is selected from the group consisting of:


59. The method of claim 31, wherein: the first reagent is represented bystructural formula IId:

and the second reagent is represented by structural formula IIIe:

whereby the compound is represented by structural formula Ig:

wherein: Y′ is methylene or a bond; X³ is —NR³R³′ or optionallyhalogenated phenyl; and R^(a), R³′, and R¹ are independently —H, C₁₋₆alkyl, C₁₋₆ alkanoyl or C₁₋₆ aralkanoyl, or —R¹ represents a lone pairof the nitrogen to which it is attached; Z is a β-bonded alkyl oralkenyl linking group, whereby Ring C is a heterocyclyl or heteroarylring; and Ring C is optionally substituted at any substitutable ringatom with halogen, —CN, —NO₂, —R^(a), —OR^(a), —N(R^(a)R^(b)), ═O, orphenyl.
 60. The method of claim 54, wherein the compound is selectedfrom the group consisting of:


61. The method of claim 54, wherein the compound is selected from thegroup consisting of:


62. The method of claim 31, wherein: the first reagent is represented bystructural formula IIe:

the second reagent is represented by structural formula IIIf:

whereby the compound is represented by structural formula Ih:

wherein X¹′ is —SH and X²′ is ═NR², or X¹′ is ═S and X²′ is —NHR²; W′ is—Cl, —Br, or —I; R³, R³′, R^(y) and R² are independently —H, —OH, C₁₋₆alkyl, C₁₋₆ alkoxy, or C₁₋₆ aralkyl, provided that at least two ofR^(a), R³′, R¹ and R² are —H; or R³ and R² together are —N═CH— or—CH═N—, optionally substituted with —N(R^(a)R^(b)) or—C(O)N(R^(a)R^(b)); the ring represented by Ring A is: an optionallysubstituted naphthyl, pyridyl, quinolinyl, or isoquinolinyl; or a phenylthat is monsubstituted at its 2, 3, or 4 positions or disubstituted atits 2,3, 2,4, or 2,5 or 3,4 positions, wherein the 1 position is thepoint of attachment of Ring A to the rest of the compound; andsubstituents for Ring A are independently —Br, —Cl, —F, —CN, —NO₂, C₁₋₆alkoxy, methylenedioxy or —CF₃, provided that when Ring A is phenyl atleast one substituent of Ring A is —F or —Cl.
 63. The method of claim31, wherein the first reagent is prepared from a third reagentrepresented by structural formula IV:

by converting the hydroxyl group bound to Y in the third reagent to —NH₂or a leaving group selected from halogen, optionally alkylated haloniumion, optionally alkylated oxonium ion, perchlorate,amonioalkanesulfonate, halosulfonate, haloalkyl sulfonate, alkylsulfonate, optionally substituted aryl sulfonate, or optionallysubstituted —N(arylsulfonate)₂.
 64. The method of claim 62, furthercomprising preparing the third reagent by reacting a fourth reagentrepresented by structural formula Va:

with R^(y)′MgCl, R^(y)′MgBr, R^(y)′MgI, R^(y)′Li, R^(y)′Na, or R^(y)′K,wherein R^(y)′ is C₁₋₆ alkyl, C₁₋₆ aralkyl, or aryl.
 65. The method ofclaim 62, further comprising preparing the third reagent by reducing afifth reagent represented by structural formula Vb:


66. A method of inhibiting proliferation of a cell, comprisingcontacting the cell with an effective amount of the compound of claim 1.67. The method of claim 66, wherein the regulation of proliferation inthe cell is mediated by at least one protein selected from the groupconsisting of retinoblastoma tumor suppressor protein andserine-threonine kinase Raf-1.
 68. A method of modulating Rb:Raf-1binding in a proliferating cell, comprising contacting the cell with aneffective amount of the compound of claim
 1. 69. The method of any oneof claims 66-68, wherein the cell is in vivo.
 70. A method of modulatingthe Rb:Raf-1 interaction in a proliferating cell, comprising contactingthe cell with a modulator of the Rb:Raf-1 interaction that is suitablefor oral administration.
 71. The method of claim 70, wherein the cell isin vivo.
 72. The method of claim 71, wherein the modulator of theRb:Raf-1 interaction is orally administered.
 73. A pharmaceuticalcomposition comprising the compound of claim 1 or a pharmaceuticallyacceptable salt or solvate thereof.
 74. A method of treating orameliorating a cell proliferation disorder, comprising contacting theproliferating cells with an effective amount of the compound of claim 1.75. The method of claim 74, wherein the regulation of proliferation inthe cells is mediated by at least one protein selected from the groupconsisting of retinoblastoma tumor suppressor protein andserine-threonine kinase Raf-1.
 76. The method of claim 75, wherein thecell proliferation disorder is cancer or a non-cancerous cellproliferation disorder.
 77. The method of claim 76, wherein theregulation of proliferation in the cells is mediated by the interactionbetween retinoblastoma tumor suppressor protein and serine-threoninekinase Raf-1.
 78. The method of claim 77, wherein the cell proliferationdisorder is a cancer selected from the group consisting of fibrosarcoma,myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma,angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer,breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma,basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceousgland carcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testiculartumor, lung carcinoma, small cell lung carcinoma, non-small cell lungcarcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma,melanoma, neuroblastoma, retinoblastoma, acute lymphocytic leukemia,lymphocytic leukemia, large granular lymphocytic leukemia, acutemyelocytic leukemia, chronic leukemia, polycythemia vera, Hodgkin'slymphoma, non-Hodgkin's lymphoma, multiple myeloma, Waldenstrobm'smacroglobulinemia, heavy chain disease, lymphoblastic leukemia, T-cellleukemia, T-lymphocytic leukemia, T-lymphoblastic leukemia, B cellleukemia, B-lymphocytic leukemia, mixed cell leukemias, myeloidleukemias, myelocytic leukemia, myelogenous leukemia, neutrophilicleukemia, eosinophilic leukemia, monocytic leukemia, myelomonocyticleukemia, Naegeli-type myeloid leukemia, nonlymphocytic leukemia,osteosarcoma, promyelocytic leukemia, non-small cell lung cancer,epithelial lung carcinoma, pancreatic carcinoma, pancreatic ductaladenocarcinoma, glioblastoma, metastatic breast cancer, melanoma, andprostate cancer.
 79. The method of claim 77, wherein the cellproliferation disorder is a cancer selected from the group consisting ofosteosarcoma, promyelocytic leukemia, non-small cell lung cancer,epithelial lung carcinoma, pancreatic carcinoma, pancreatic ductaladenocarcinoma, glioblastoma, metastatic breast cancer, melanoma, andprostate cancer.
 80. The method of claim 74, wherein the cellproliferation disorder is angiogenesis or the cell proliferationdisorder is mediated by angiogenesis.
 81. The method of claim 74,further comprising administering an anticancer drug.
 82. A method ofinhibiting angiogenic tubule formation in a subject in need thereof,comprising administering to the subject an effective amount of thecompound of claim
 1. 83. A method of treating or ameliorating a cellproliferation disorder, comprising contacting proliferating cells withan effective amount of the compound of claim 1, wherein theproliferating cells have an elevated level of Rb, Raf-1, or Rb bound toRaf-1.
 84. The method of claim 66, wherein the cells have an elevatedlevel of Rb, Raf-1, or Rb bound to Raf-1.
 85. The method of claim 66,further comprising assaying the level of Rb, Raf-1, or Rb bound to Raf-1in the cell.
 86. A method of assessing a subject for treatment with aninhibitor of Rb:Raf-1 binding interactions, comprising determining, inthe subject or in a sample from the subject, a level of Rb, Raf-1, or Rbbound to Raf-1, wherein treatment with an inhibitor of Rb:Raf-1 bindinginteractions is indicated when the level of Rb, Raf-1, or Rb bound toRaf-1 is elevated compared to normal.
 87. The method of claim 86,wherein the inhibitor of RB:Raf-1 binding interactions is the compoundof claim
 1. 88. A method of identifying a subject for therapy,comprising: providing a sample from the subject; determining a level ofRb, Raf-1, or Rb bound to Raf-1 in the sample; and identifying thesubject for therapy with an inhibitor of Rb:Raf-1 binding interactionswhen the level of Rb, Raf-1, or Rb bound to Raf-1 is elevated comparedto normal.
 89. The method of claim 88, wherein the inhibitor of Rb:Raf-1binding interactions is the compound of claim
 1. 90. A kit, comprising:an antibody specific for Rb, Raf-1, or Rb bound to Raf-1; andinstructions for determining the level of Rb, Raf-1, or Rb bound toRaf-1 in a sample using the antibody specific for Rb, Raf-1, or Rb boundto Raf-1.